Enhancing chromatographic separation of components in a mixture by employing a decreasing gradient of complexing agent

Abstract

A method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material, the method comprising the following steps: - loading the ion exchange material with a loading buffer containing the drug delivery carrier mixture and a complexing agent; - rinsing the ion exchange material with an elution buffer containing a complexing agent at a lower concentration than that in the loading buffer or without a complexing agent; - thereby eluting the drug delivery carrier from the ion exchange material through the elution buffer; and optionally, collecting the separated drug delivery carrier.

Description

Technical Field

[0001] This invention relates to a chromatographic method that utilizes ion exchange materials to separate a mixture of drug delivery carriers in a buffered aqueous mixture. Background Technology

[0002] In complex biological mixtures, there is always a need to improve the separation of target components, especially when byproducts are involved (such as biomolecules like proteins and nucleic acids, or even more complex biological materials like viral particles, intact cells, or cellular components). Removing all impurities is one of the most challenging steps in the production of biomolecules or the isolation of effective vectors. The most concerning issue is the potential for serious side effects from these impurities. To achieve high potency and safety for target biomolecules, these impurities need to be characterized, detected, and ultimately removed during their production.

[0003] Most methods currently on the market are difficult to scale up, which is a major drawback in preparative purification.

[0004] Methods for specifically detecting, identifying, and separating impurities from complex mixtures of contaminants and pollutants exhibiting similar properties to the target substance also present challenges. rAAVs are an example of this behavior, where only minute charge differences exist between empty, fully filled, and partially filled capsids. Fully filled capsids are considered to be the same size as empty capsids, but their isoelectric point (pI) is slightly lower (within 0.4 pH units). Therefore, removing unwanted contaminants and impurities using conventional methods often results not only in a loss of target substance yield but also in overlap of the target biomolecule with other impurities of similar electronegativity.

[0005] Chromatography has proven to be a powerful tool for separating, purifying, and extracting target substances in production processes. In particular, small molecules can be purified using different types of chromatographic methods, such as ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, and others.

[0006] The conventional method for eluting target substances in chromatography is to use an increasing salt gradient, usually in a linear mode.

[0007] When the target substance (e.g., protein) is negatively charged, anion exchange chromatography (AEX) is used. When chromatography is performed at a pH above the protein's isoelectric point (pI), the protein will become negatively charged. In this type of chromatography, the stationary phase is positively charged, and the protein is adsorbed after negatively charged molecules are loaded. Elution of the target substance (e.g., protein) is primarily achieved using a high-salt-concentration elution buffer. The target substance is collected in the high-salt-concentration fraction. This condition is sometimes unfavorable because the target substance may be impaired due to partial or complete denaturation. Another disadvantage is the need to remove the high salt concentration from the target substance in downstream steps via dialysis, tangential flow filtration, or other separation methods, which can lead to a loss of target substance yield due to the applied high shear forces.

[0008] While AEX chromatography is a powerful tool in many manufacturing processes, it can sometimes reach its limits when purifying and separating complex samples as described above, especially when using increasing salt concentrations in the elution buffer.

[0009] Purpose of the invention One object of the present invention is to provide a method that can overcome the shortcomings of existing ion exchange chromatography methods.

[0010] Another objective is to provide a method for isolating, extracting and purifying multiple subgroups or subtypes of various biomolecules, such as viruses, viral and lipid nanoparticles, extracellular vesicles, proteins, nucleic acids and so on.

[0011] Another object of the present invention is to provide a method for separating a mixture of contaminants and a pharmaceutical delivery carrier in a buffered aqueous mixture.

[0012] Another object of the present invention is to provide a method for purifying a pharmaceutical delivery carrier mixture in a buffered aqueous mixture.

[0013] A further objective of this invention is to provide a method for separating a mixture of drug delivery carriers in a buffered aqueous mixture using binary, ternary, or multiple anion and / or cation exchange gradient conditions. This method is capable of separating multiple subgroups or subtypes of different biomolecules (such as viruses, bacteriophages, viral and lipid nanoparticles, extracellular vesicles, proteins, nucleic acids, etc.).

[0014] Another object of the present invention is to provide a method for separating a pharmaceutical delivery carrier mixture in a buffered aqueous mixture, which can be used on both analytical and preparative scales. Summary of the Invention

[0015] The objective of this invention is achieved through a method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material. This method includes the following steps: The ion exchange material is loaded with a loading buffer containing the drug delivery carrier mixture and a complexing agent, and then... The ion exchange material is rinsed with a solution containing a lower concentration of complexing agent than that in the loading buffer, or without a complexing agent. This elutes the drug delivery carrier from the ion exchange material, and optionally... Collect the separated drug delivery carriers.

[0016] In another definition, the present invention is a method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material, the method comprising the following steps: The ion exchange material is loaded with a loading buffer containing the drug delivery carrier mixture and free of complexing agents, and then... The ion exchange material was rinsed with a washing buffer containing a complexing agent. The drug delivery carrier is eluted from the ion exchange material using an elution buffer containing a lower concentration of a complexing agent than the washing buffer, or without a complexing agent, and optionally... Collect the separated drug delivery carriers.

[0017] In a further definition, this invention is a method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using ion exchange materials, the method comprising the following steps: The ion exchange material is loaded with a loading buffer containing the drug delivery carrier mixture, and then... The ion exchange material was rinsed with a washing buffer. The loading buffer or the washing buffer, or both, contain a complexing agent. The drug delivery carrier is eluted from the ion exchange material using an elution buffer, employing a salt gradient or pH gradient, wherein the elution buffer contains a lower concentration of a complexing agent than the loading buffer or the elution buffer, or contains no complexing agent, and optionally... Collect the separated drug delivery carriers.

[0018] This invention provides a chromatographic method that utilizes complexing agents of at least divalent, trivalent, tetravalent or higher valence states to achieve advantageous separation of multiple subgroups or subtypes of different biomolecules (e.g., viruses, bacteriophages, viral and lipid nanoparticles, extracellular vesicles, proteins, nucleic acids, etc.) under decreasing concentration (gradient) conditions.

[0019] The complexing agent may already be present in the buffered aqueous mixture containing the drug delivery carrier, or it may be present at the start of loading the ion exchange material. The complexing agent may be present in the loading buffer or in the buffer in which the sample contacts the ion exchange chromatographic material. In a particular embodiment of the method of the present invention, the sample is diluted in a loading buffer containing the complexing agent. Typically, the ion exchange material is equilibrated with the loading buffer before loading the sample (especially a sample dissolved in the loading buffer).

[0020] In some embodiments of the method of the present invention, the sample may be prepared and loaded onto IEX chromatographic material using a loading buffer without a complexing agent. Subsequently, a complexing agent is introduced after the sample loading step.

[0021] In one embodiment of the invention, the delivery vector may be selected from the group consisting of viruses, virus-like particles, extracellular vesicles, lipid nanoparticles, or combinations thereof. Specifically, the virus may be parvovirus, adeno-associated virus (AAV), for example, selected from the following serotypes of AAV: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, AAV12; mixed serotypes, such as recombinant mixed serotypes like AAV2 / 8; AAV chimeras, surface-modified AAVs, or synthetically derived AAV-like particles.

[0022] In another embodiment of the present invention, the delivery vector may be an AAV capsid, a lentiviral capsid, an adenovirus, or the extracellular vesicle may be an exosome or a bacteriophage, or a combination thereof.

[0023] In another embodiment of the invention, the pharmaceutical preparation delivered by the pharmaceutical preparation delivery carrier can be any substance with pharmaceutical activity, particularly genetic material such as DNA or RNA, or protein materials such as antibodies, or non-biological materials.

[0024] In another embodiment of the invention, the ion exchange material may be an anion exchanger, a cation exchanger, or a combination thereof. Specifically, the ion exchange material may be a mixed-mode chromatographic material, a monolithic column anion or cation exchanger, a monolithic column multimode material, a particulate anion or cation exchanger, and / or a multimode material.

[0025] In another embodiment of the invention, the ion exchange material may be mounted on a membrane, mounted in the form of particles in a column, and / or mounted in a fiber column.

[0026] The complexing agent is an organic compound.

[0027] In one embodiment of the invention, the complexing agent is at least divalent, preferably trivalent, tetravalent, or higher. The complexing agent may contain at least three chemical groups capable of providing a negative charge in an aqueous medium. Typical examples of such chemical groups are those capable of providing anionic groups, particularly carboxyl groups. Another term for these compounds is chelating agent.

[0028] In one embodiment, the complexing agent comprises at least three chemical groups capable of providing a positive charge in an aqueous medium, such as protonated amine groups.

[0029] For example, the complexing agent may be selected from the group consisting of ethylenediaminetetraacetic acid, citric acid, pyrophosphate, diethyltriaminepentaacetic acid, tetrasodium glutamate diacetate, ethylene glycol-bis(oxyethyleneimino)tetraacetic acid, cyclohexanediaminetetraacetic acid, and combinations thereof.

[0030] It can also be a polyamine salt, preferably selected from the group consisting of spermine, spermidine, and combinations thereof.

[0031] In another embodiment of the invention, the elution buffer may contain an increasing salt concentration gradient and / or pH gradient. The elution buffer may contain a different pH value or a moderately high salt concentration than the loading buffer to elute empty capsids and partially filled capsids, respectively.

[0032] A gradient refers to the systematic alteration of the mobile phase composition during separation to enhance the elution of compounds. Typically, a gradient begins with "weak" elution conditions that retain the compound on the material and gradually transitions to "strong" conditions that promote elution. This method improves separation efficiency by allowing different compounds to elute at different times, thereby enhancing resolution and peak capacity. Common gradient types include linear gradients, isocratic (constant conditions), and step gradients. Linear gradients are preferred.

[0033] A pH gradient can be either an increasing or decreasing pH gradient.

[0034] The elution buffer may optionally or in combination initially contain a salt with low elution properties, which is then replaced by a salt with high elution properties during elution.

[0035] When using a buffer with a moderately high salt concentration or a moderate pH gradient relative to the loading buffer, it is preferable to elute and separate empty and partially filled capsids, which reflects classical ion exchange chromatography.

[0036] As described in this invention, elution of primarily full capsids and other capsids (which may be heavy capsids, aggregates, or capsid fragments) can be achieved by using a high-salt-concentration buffer of the same salt (optionally, a more eluting salt can be used, or even the pH can be changed during elution). By changing the salt conditions of the elution buffer using a more eluting salt, a high salt load (which may be necessary in other cases) can be avoided.

[0037] After initial elution with a buffer with a moderate salt concentration, it may be advantageous to change the elution conditions and perform subsequent elutions with a buffer containing a salt with a higher salt concentration and greater elution capacity and / or a buffer with a different pH value.

[0038] In another embodiment of the invention, the elution solution may contain at least one stabilizer. Typically, the stabilizer may be selected from the group consisting of amino acids (particularly arginine, lysine, and histidine), choline, glycopeptides, nucleotides, sugars, and combinations thereof. The function of the stabilizer is to protect the delivery carrier from stress damage (heat, shear force, etc.) during processing.

[0039] In another embodiment of the invention, the pH of the elution solution may be 3 to 10, or 7 to 10, particularly 9 to 10, or 9.75.

[0040] In another embodiment of the invention, the concentration gradient of the complexing agent in the elution solution can be as low as possible to achieve the binding conditions and the desired separation. Typically, the complexing agent concentration used in the loading or rinsing steps of the method of the present invention is from 2 mM to 0.25 mM, particularly from 1.5 mM to 0.5 mM. Compared to, for example, NTA, multivalent complexing agents (such as EDTA) exhibit higher potency, allowing them to be used at lower concentrations.

[0041] In another embodiment of the invention, the elution buffer may comprise an alkaline pH, at least one ionizable salt, at least one buffering agent, at least one isotonic substance, at least one auxiliary agent, and / or at least one organic modifier. Typically, the isotonic substance may be selected from the group consisting of sucrose, sorbitol, mannitol, xylitol, and mixtures thereof; the auxiliary agent may be a nonionic surfactant, such as poloxamer 188 or Tween. ® 20. Tween ® 80. Tergitol ® Triton X-100, etc. Preferably, the organic modifier may be selected from the group consisting of: acetonitrile, 1-butanol, tert-butanol, propylene carbonate, isopropanol, ethanol, methanol, ethylene glycol, 1-propanol and mixtures thereof. Attached Figure Description

[0042] Figure 1 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separation in filled / empty AAV9 capsids under a combination of decreasing gradients of the complexing agent and increasing gradients of a single linear salt (0 mM to 200 mM NaCl) as commonly used.

[0043] Figure 2 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separation in filled / empty AAV9 capsids under a combination of decreasing complexing gradients and two increasing salt gradients.

[0044] Figure 3 A chromatogram is shown, illustrating the tryptophan fluorescence resolution of AAV9 capsid separation in full / empty capsids when using buffers with or without arginine as AAV9 capsid stabilizers, under a combination of decreasing complexing gradients and two increasing salt gradients.

[0045] Figure 4 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separation in filled / empty AAV9 capsids under various combinations of complexing agent gradients and two incremental salt gradients.

[0046] Figure 5 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separation in filled / empty AAV9 capsids under a combination of a decreasing complexing gradient and two increasing ionization gradients.

[0047] Figure 6 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separation in filled / empty AAV9 capsids when using different disodium complexing agents (except trisodium citrate).

[0048] Figure 7 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separated from the filled / empty AAV9 capsid using disodium and a fully deprotonated complexing agent.

[0049] Figure 8 A chromatogram is shown, illustrating the fluorescence resolution of tryptophan separated from the filled / empty AAV9 capsid using a two-dimensional chromatographic system.

[0050] Figure 9 A chromatogram is shown, illustrating the UV resolution of the separation of fully / partially filled / empty AAV9 capsids using this method, with the sample prepared and loaded without the addition of a complexing agent. Detailed Implementation

[0051] The term “resolution” is known to those skilled in the art. Resolution is calculated using the following formula [1] by dividing the difference in retention time between different chromatographic peaks by the peak width at half maximum of each peak.

[0052] R=1.18 (t R2 -t R1 ) / (W h1 +W h2 ), where t R2 >t R1 t R1 , t R2 Retention time for peak W h1 W h2 The peak width at half height The term "full AAV capsid" refers to a capsid containing a sufficient amount of vector genome to produce a therapeutic effect.

[0053] The term "empty AAV capsid" refers to a capsid that lacks sufficient vector genome and therefore cannot provide therapeutic benefits.

[0054] When temperature is mentioned or not mentioned, the temperature is room temperature (23°C).

[0055] When volume is mentioned without specifying temperature, the volume is the volume at room temperature.

[0056] In the following description, the separation method as described in this invention will sometimes be described in detail by way of example with reference to the separation of empty and full AAV capsids. This is not intended to limit the invention to the separation of empty and full AAV capsids.

[0057] Conventionally, after the drug delivery carrier mixture is contacted with the stationary phase (e.g., by loading the mixture onto ion-exchange chromatographic material), separation of the delivery carrier is achieved by treating the chromatographic material with a gradient buffer. This gradient can be formed by increasing the salt concentration of the buffer to enhance its ionic strength. Alternatively, a gradient can be formed by altering the pH of the buffer in contact with the chromatographic material.

[0058] However, as described in this invention, the separation of drug delivery carriers (e.g., empty and partially filled AAV capsids) is preferably achieved by simultaneously reducing the gradient of the complexing agent (e.g., EDTA, pyrophosphate, citrate, or mixtures thereof) and employing an intermediately increasing gradient of a first salt (e.g., a monovalent or divalent salt) in the elution buffer. The salt concentration range can be from 50 mM to 250 mM, and for AAV9, particularly 60 mM to 70 mM or 65 mM.

[0059] Separation of other capsids (e.g., the primary desired capsids, those fully loaded with drug reagents, such as those fully loaded with AAV9) was achieved by further increasing the salt concentration gradient in the elution buffer (e.g., increasing it to 2000 mM).

[0060] An increasing elution gradient can also be generated in the elution buffer. In this example, the elution buffer initially contains a salt with lower elution properties, which is replaced by a salt with higher elution properties during elution. The term “elution property” refers to the so-called Hofmeister sequence (elution property increasing) [2].

[0061] For example, when performing anion exchange chromatography using the method of the present invention, the following ions (from low to high ionization) are preferred: Anion: CH3CH2COO - <CH3COO - <HCOO - <F - <C - <Br - - <ClO4 - <SCN - Cation: N(CH3)4 + <NH4 + <Cs + <Rb + <K + <Na + <Li + <Mg 2+ <Ca 2+ <Zn 2+ <Ba 2+ .

[0062] By using salts with different elution properties, such as sodium perchlorate in the first part of elution and calcium perchlorate in the second part of elution, the increase in salt concentration can be largely avoided.

[0063] Those skilled in the art tend to maintain the lowest possible salt concentration to prevent aggregation or subsequent sample desalting, yet it needs to be high enough to achieve good separation of the drug carrier. To balance these requirements, those skilled in the art can easily find suitable conditions without the burden of excessive experimentation.

[0064] When only a single linear gradient is conventionally used, the separation effect between different types of drug delivery vehicles (e.g., empty capsids, fully capsids, partially filled capsids, damaged capsids, aggregated capsids, and similarly obtained vehicles) is poor or nonexistent. Figure 1 ).

[0065] If the separated drug formulation carrier is to be applied subsequently, it is even more ideal to reduce the gradient of the complexing agent in order to obtain a safer storage medium (e.g., a final full AAV9 capsid), as complexing agents (such as EDTA) may be harmful to health. For this reason, the concentration of the complexing agent should be as low as possible to enhance AAV9 capsid separation.​

[0066] Figure 5 Baseline separation between empty and full capsids is shown. Only the loading buffer contained a 1 mM Na2-EDTA complexing agent. The intact capsid was eluted using a buffer with the same salt concentration but increasing elution properties. With this elution method, the complexing agent concentration in the final full AAV9 fraction was removed during the elution process.

[0067] In one embodiment of the present invention, the method includes steps (i) to (vi): (i) Provide an aqueous mixture of the components, (ii) Contact the aqueous mixture with the chromatographic material. (iii) Separation is then initiated by contacting the chromatographic material with a buffer containing a complexing agent. (iv) Subsequently, the salt concentration is increased to create a salt concentration gradient, or an ionization gradient is created by using another salt with higher ionization properties that does not contain a complexing agent to reduce the complexing agent concentration, thus forming a decreasing complexing agent gradient. (v) Collect the components that may have separated due to de-complexation, mainly empty capsids and partially filled capsids. (vi) The salt gradient is then increased further, or alternatively, the salt is replaced with another salt with higher elution properties to wash away the remaining capsids, mainly full capsids, overfilled capsids and aggregates.

[0068] Specifically, the virus may be: (i) parvovirus, (ii) Adeno-associated viruses (AAVs), particularly those selected from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, and AAV12. (iii) Mixed serotypes, especially recombinant mixed serotypes, such as AAV2 / 8, (iv) AAV chimera, (v) Surface-modified AAV, and (vi) Synthetic derivative AAV-like particles.

[0069] In another embodiment of the present invention, the ion exchange material may be: (i) Mixed-mode chromatographic materials, (ii) Monolithic column anion exchanger. (iii) Monolithic column cation exchanger, (iv) Multi-mode materials for monolithic columns, (v) Particulate anion exchangers, and / or (vi) Multimodal materials.

[0070] In another embodiment of the invention, the chromatographic material may be mounted on a membrane, in the form of particles in a column, and / or in a fiber column.

[0071] In another embodiment of the invention, the aqueous environment may be an alkaline pH aqueous solution containing a complexing agent, a liquid-free salt, a buffer, an isotonic substance, an auxiliary agent, and / or an organic modifier, suitable for QA column or AEX chromatography. Typically, the buffer solution has a pH higher than 9.5, preferably 9.75.

[0072] In another embodiment of the invention, the isotonic substance may be selected from sucrose, sorbitol, mannitol, xylitol and mixtures thereof.

[0073] In another embodiment of the present invention, the adjuvant preparation may be a nonionic surfactant, such as poloxamer 188 or Tween. ® 20. Tween ® 80. Tergitol ® Triton X-100, etc. The organic modifier may be selected from the following group: acetonitrile, 1-butanol, tert-butanol, propylene carbonate, isopropanol, ethanol, methanol, ethylene glycol, 1-propanol and mixtures thereof.

[0074] In another embodiment of the invention, the adjuvant preparation may also be a stabilizer, such as amino acids (especially basic amino acids, such as arginine or lysine, histidine), choline, glycopeptides, or nucleotides.

[0075] For example, the ion exchange material is a strong or weak anion exchanger. The anion exchange material may exhibit hydrogen bonding properties or may be compounded with a positively charged metal affinity ligand as an example of a multimode material. The anion exchanger may also be a monolithic column anion exchanger or multimode material, a particulate anion exchanger or multimode material, and / or anion exchanger or multimode material mounted on a membrane, and / or a particle-filled anion exchange column or multimode column, and / or a fiber chromatography anion exchanger or multimode fiber column. Strong anion exchange materials contain quaternary ammonium ligands, commercially known as Q, QA, QAE, QAM, TEAE, TMAM, or TMAE, with a consistent charge in the pH range of approximately 2 to 13. Methods for using quaternary ammonium anion exchange materials to separate empty and full caps have been described [3].

[0076] The weak anion exchanger DEAE (diethylaminoethyl) material contains a tertiary amine ligand with a pKa of approximately 11.5, and can elute empty and full caps at moderate pH values ​​[4].

[0077] Multimode chromatography, also known as mixed-mode chromatography (MMC), refers to chromatographic methods that utilize more than one form of interaction between the stationary phase and the analyte to achieve their separation [5, 6, 7]. On one hand, MMC can be considered a subtype of AEX. MMC can be divided into physical MMC and chemical MMC. In the former, the stationary phase consists of two or more types of packing materials. In the chemical method, a packing material containing two or more functional groups is used. One method is to connect two commercial columns in series, called a "tandem column." Another method is a "two-phase column," which is made by packing two stationary phases separately at both ends of the same column. Yet another method is to uniformly mix two or more different types of stationary phases in a single column, which is called a "mixed column" or "mixed-bed column."

[0078] Monolithic column anion exchange materials are particularly well-suited for performing the methods of this invention. The experiments described in the "Examples" section were conducted on such materials.

[0079] Similarly, other materials provided in the form of particulate ion exchange materials, and / or ion exchange materials mounted on membranes, and / or particulate-filled ion exchange columns, and / or fiber chromatography ion exchangers or multimode fiber columns are also suitable. Ion exchange materials include anion exchangers and cation exchangers.

[0080] The material can be a multimode metal affinity exchange material. This material combines the properties of positively charged metal affinity ligands with those of a weak anion exchanger with hydrogen bonding characteristics [8]. It can first separate empty capsids in a linear magnesium chloride gradient, then separate full capsids, and finally elute the vast majority of empty capsids in a subsequent high-salt step.

[0081] The separation, purification, or extraction of the drug delivery carrier is carried out in a substantially aqueous environment. This environment contains the sample to be separated and contaminants. Contaminants may be impurities or pollutants generated during the production of the target product.

[0082] For AAVs loaded with genetic material for gene therapy purposes, several unwanted substances exist that must be removed before administration to patients. For example, in addition to the required loaded AAV (full AAV), there are empty AAV capsids that must be removed. Furthermore, contaminants such as AAV aggregates or partially loaded AAV may be present in the production batch. Needless to say, these contaminants or impurities must be removed.

[0083] For simplicity, the present invention will be described in detail below by using anion exchange chromatography materials to separate empty AAV capsids from full AAV capsids.

[0084] The process of removing unwanted substances and separating empty and full AAV capsids begins by providing a mixture from the AAV capsid production process in an aqueous environment. The mixture (optionally dissolved in a loading buffer containing a complexing agent such as EDTA) is brought into contact with the chromatographic material by loading it into a column containing the chromatographic material. Monolithic column anion exchangers are particularly useful.

[0085] Figure 1 The effect of a combination of conventionally used increasing salt gradients and decreasing complexing agent gradients on AAV9 capsid separation is demonstrated. When using a combination of increasing salt gradients and decreasing complexing agent gradients, the resolution between empty and full AAV9 capsids is poor. When two different salt gradients are introduced (one with a lower elution salt concentration (1) and the other with a higher concentration of the same elution salt than the first elution salt (1) (2)), the resolution between empty and full capsids is improved. Figure 2 This invention demonstrates improved AAV9 capsid separation. The method operates at a relatively high pH (optimal pH 9.75; no similar effect was observed at pH 9.5). To stabilize the AAV9 capsid, 50 mM arginine was added to the buffer. Even without arginine, AAV9 separation remained effective, with only a slight increase in peak size after the full AAV9 capsid peak, which was considered to be due to aggregates or damaged capsids. Figure 3 ).

[0086] Complexing agents, such as EDTA, are generally considered potentially hazardous. In this sense, decreasing the complexing agent gradient and ensuring its lowest possible concentration in the final AAV9 product seems like a sensible precaution. Therefore, to validate the concept of the method of this invention, several different buffer combinations were tested ( Figure 4 ).

[0087] Figure 4 The resolution of AAV9 capsid separation is demonstrated when using buffers with and without Na2-EDTA. Adding Na2-EDTA only to buffer A improves the separation of the AAV9 capsid.

[0088] Samples were also eluted by increasing the elution properties of the buffer while maintaining the same salt concentration, using buffers B and C ( Figure 5 Compared to buffer C, buffer B has lower elution properties. By doing so, fully capped AAV9 capsids are eluted at a relatively low salt concentration (buffer C is 65 mM calcium perchlorate) compared to the high-salt wash required for capsid elution using the first method. Even lower concentrations of calcium perchlorate successfully eluted all fully capped AAV9 capsids.

[0089] like Figure 5 The components were collected as shown.

[0090] Orthogonal analysis—dPCR and mass spectrometry—was performed on the collected components, and the results confirmed that eluent 3 contained the vast majority of AAV9 capsids ( Figure 5 (Table 1).

[0091] Table 1: Orthogonal analysis: dPCR (average concentration in vg / mL) and mass spectrometry (percentage of AAV capsid). Different complexing agents were tested to determine the optimal candidate. Figure 6 The separation effects of AAV9 capsids using different disodium complexing agents (except trisodium citrate) are demonstrated. In this series, the best separation results were obtained when using trivalent or tetravalent carboxyl complexing agents. The complete deprotonation of the complexing agent slightly altered the percentage of fully filled AAV9 capsids and the spectra of empty and partially filled AAV9 capsids. Figure 7 ).

[0092] The method of this invention is also applicable to the analysis of AAV9 lysis buffer samples. Two-dimensional chromatography system PATfix ® AAVSwitcher (Sardolibia Separation Company, Aidovschina, Slovenia) enables the analysis of complex lysate samples upstream of process development. The first column, a strong cation exchange column, is used for sample purification; the second column, an anion exchange column, separates empty, partially filled, and fully filled capsids.

[0093] like Figure 8 As shown, this new method enables the separation of empty, partially filled, and fully filled AAV9 capsids in purified samples. The figure illustrates the fluorescence response of the separated AAV9 capsids on an anion exchange column via a decreasing gradient of the complexing agent (Na2-EDTA) and an increasing gradient of the ionizing salt in buffer A.

[0094] Figure 9 This demonstrates a method for sample preparation and loading buffers that do not contain a complexing agent. The complexing agent is introduced into the column in the next step, followed by an increasing salt gradient applied to the column, resulting in a corresponding decrease in the complexing agent concentration.

[0095] Example Example 1A: Preparation of AAV9 capsid for method development and orthogonal analysis rAAV9 was generated via triple plasmid transfection of the suspension HEK293 cell line. Rep2-Cap9 and Helper plasmids were used, along with a cis-construct of a GFP expression cassette containing AAV2 inverted terminal repeat (ITR) regions flanked by these regions. The plasmids were mixed at a molar ratio of 1:1:1 and treated with PEI MAX.® Transfection reagent (Polysciences, Warrington, Pennsylvania, USA) was used to transfect cells. Transfection was performed in a 5L stirred tank Biostat® B-DCU bioreactor (Sartorius, Göttingen, Germany) using a fed-batch method. 72 hours post-transfection, the surfactant Tween was added directly to the bioreactor. ® Cell lysis was performed using a Sigma-Aldrich 20 (SQA) microscope. Materials were collected and frozen at -80°C until further use. The lysed AAV9 serotype was clarified and then processed using a TFF pre-capture step coupled with DNase treatment. A cation exchange column, CIMmultus, was used. TM SO3 (Sardolibia Separation Company, Aidovschina, Slovenia) was used to capture the sample and further purify it. Sartorius was used. ® The Vivaspin Turbo 100kDa PES concentrator (Sartorius, Göttingen, Germany) replaces the eluent from the capture step with a formulation buffer. Samples prepared in this way can be used for the development of chromatographic separation methods and orthogonal analysis.

[0096] Example 1B: Preparation of AAV9 capsid for method development and orthogonal analysis Sample generation was the same as in Example 1A, except that density gradient ultracentrifugation (DGUC) was used for purification. In DGUC, the capsid population is separated according to its density. Full capsids sink to the bottom, while empty capsids are fractionated at the top of the CsCl gradient. Partially filled capsids lie between the top and bottom of the CsCl gradient.

[0097] Example 2: Enhancing AAV9 capsid separation by a decreasing complexing agent gradient and an increasing salt gradient Use the following buffer: Buffer A: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 200 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TMThe analysis and separation of empty and full AAV9 capsids were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A) containing 1 mM Na2-EDTA complexing agent, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. Empty and full AAV9 capsids in the samples were eluted using a 160 CV linear salt gradient (elution buffer, Buffer B): the elution buffer contained 200 mM sodium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The flow rate was 1 mL / min.

[0098] like Figure 1 As shown, the corresponding buffer combination resulted in poor resolution (1.06) between empty and full AAV9 capsids. The peaks corresponding to full AAV9 capsids were relatively broader compared to those corresponding to empty AAV9 capsids.

[0099] Example 3: Enhancing AAV9 capsid separation by decreasing complexing agent gradient and increasing salt gradient Use the following buffer: Buffer A: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 50 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer C: 20 mM BTP + 50 mM arginine + 300 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TMThe analysis and separation of empty and full AAV9 capsid samples were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A), which consisted of a buffer containing 1 mM Na2-EDTA complexing agent, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, at pH 9.75. The majority of empty capsids and partially filled AAV capsids were eluted using a 40 CV linear salt gradient (elution buffer, Buffer B): the elution buffer contained 50 mM sodium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, at pH 9.75. The flow rate was 1 mL / min. A 10 CV period was then maintained, followed by a second 40 CV linear salt gradient (elution buffer, buffer C): the elution buffer contained 300 mM sodium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, at pH 9.75. In this gradient, the vast majority of the fully capped AAV9 capsid was eluted.

[0100] like Figure 2 As shown, the corresponding buffer combination provides a high resolution of 5.46 between empty and full AAV9 capsids.

[0101] Example 4: Enhanced separation of AAV9 capsid with and without complexing agents Use the following buffer: Buffer A: 20 mM BTP + (50 mM arginine) + 1 mM Na2-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + (50 mM arginine) + 50 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer C: 20 mM BTP + (50 mM arginine) + 300 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TMThe analysis and separation of empty and full AAV9 capsid samples were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A) containing 1 mM Na2-EDTA complexing agent, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The majority of empty capsids and some of the AAV capsids in the samples were eluted using a 40 CV linear salt gradient (elution buffer, Buffer B): the elution buffer contained 50 mM sodium chloride, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. A linear salt gradient (elution buffer, buffer C) was then maintained for 10 CVs and followed by a second 40 CVs gradient: the elution buffer contained 300 mM sodium chloride, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, at pH 9.75. In this gradient, the vast majority of fully capped AAV9 capsids were eluted. The flow rate was 1 mL / min. Only 50 mM arginine was added to all three buffers (buffers A, B, and C) in one of the groups.

[0102] Compared to buffers without arginine, the corresponding buffer combinations containing arginine produced similar elution profiles. In the presence of arginine, a slightly sharper but less pronounced peak was observed after elution of the fully capped AAV9 capsid. The small, broad peaks eluted after capped peaks (retention time greater than 8 minutes) likely represent aggregates or damaged AAV9 capsids. Figure 3 ).

[0103] Example 5: Effect of complexing agents on AAV9 capsid separation Use the following buffer: Buffer A: 20 mM BTP + 50 mM arginine ± 1 mM Na2-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine ± 1 mM Na2-EDTA + 50 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 300 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TMThe analysis and separation of empty and full AAV9 capsid samples were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A), which consisted of a buffer with or without 1 mM Na2-EDTA, and also contained 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. Samples were eluted using a linear salt gradient of 40 CVs (elution buffer, Buffer B): the elution buffer contained or without 1 mM Na2-EDTA, and also contained 50 mM sodium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The flow rate was 1 mL / min. This was followed by a 10 CVs linear salt gradient (elution buffer, buffer C) and a second 40 CVs gradient: the elution buffer contained 300 mM sodium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, at pH 9.75. In this gradient, the vast majority of the fully capped AAV9 capsid was eluted.

[0104] Figure 4 The chromatograms are shown for the addition of the complexing agent Na2-EDTA to four different combinations of loading buffer (buffer A) and elution buffer (buffer B). For example, when the complexing agent is added to both buffers simultaneously, the separation between empty and full capsids is barely observable. The highest resolution of 5.46 between empty and full AAV9 capsids is achieved when the complexing agent is added only to buffer A.

[0105] Example 6: Enhancing AAV Capsule Separation by Decreasing Complexing Agent Gradient and Increasing Isolation Liquid Gradient Use the following buffer: Buffer A: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 65 mM NaClO4 + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer C: 20 mM BTP + 50 mM arginine + 65 mM Ca(ClO4)2 + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer D: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA + 2000 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TM Analytical separation of empty and full AAV9 capsid samples was performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A) containing 1 mM Na2-EDTA complexing agent, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The majority of empty capsids and partially filled AAV capsids were eluted with a linear salt gradient of 40 CVs, followed by elution with a buffer containing the first elution salt (Elution buffer, Buffer B): the elution buffer contained 65 mM sodium perchlorate, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The flow rate was 1 mL / min. Elution was then performed using a buffer containing Ca(ClO4)2 as the second eluting salt, which exhibited higher eluting properties than the first salt (eluting buffer, buffer C): the eluting buffer contained 65 mM calcium perchlorate, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75. In this gradient, the vast majority of the AAV9 capsid was eluted. Finally, the column was washed with a high-salt buffer (eluting buffer, buffer D) to remove any remaining bound material: the eluting buffer contained 2000 mM sodium chloride, 1 mM EDTA, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75.

[0106] Figure 5 The baseline separation between the empty capsid subpopulation (empty capsid 1 and empty capsid 2) and the full capsid is shown. This separation was achieved by a decreasing gradient of complexing agent (Na2-EDTA) in buffer A and an increasing gradient of elution salts (elution buffers B and C) without complexing agent.

[0107] collect Figure 5 The labeled components E1 to E5 were further analyzed using orthogonal methods (such as mass spectrometry and digital PCR). The results of the orthogonal analysis are shown in Table 1.

[0108] The molecular weight and corresponding percentage of individual capsids in components E1 to E5 were determined using mass spectrometry (Refeyn Ltd. Littlemore, Oxford, UK). Standard calibration curves were prepared using internal empty AAV capsids. Samples were diluted 10,000-fold with PBS buffer, and 20 μL of the solution was spotted onto a glass plate for scattering analysis at 488 nm.

[0109] The rAAV vector genomic titer was assessed using digital PCR (Qiagen, Hilden, Germany). Samples were pre-diluted and then treated with DNase I (New England Biolabs, Ipswich, Massachusetts, USA) to digest unpackaged DNA. Samples (fractions E1 to E5) were serially diluted 10-fold and incubated at 95°C for 10 minutes to lyse the viral capsid. Diluents matching the dPCR dynamic range were selected and transferred to 96-well nanoplates (Qiagen, Hilden, Germany) for PCR reaction and recording. Selected primers and probes for PCR were designed to align with the eGFP target sequence. dPCR cycling conditions were a two-step procedure consisting of 10 minutes at 95°C followed by 40 cycles of 15 seconds at 95°C and 30 seconds at 60°C. Detailed design, analysis, and quantification were performed according to the Qiagen dPCR User Manual (Qiagen 2021).

[0110] Example 7: Effect of different complexing agents on AAV9 capsid separation Buffer A: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA (or Na2-NTA, or Na3-citrate, or Na2-pyrophosphate) + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 60 mM NaClO4 + 1% sorbitol + 0.01% poloxamer 188; pH 9.75 Buffer C: 20 mM BTP + 50 mM arginine + 60 mM CaCl2 + 1% sorbitol + 0.01% poloxamer 188; pH 9.75 Buffer D: 20 mM BTP + 50 mM arginine + 1 mM Na2-EDTA + 2000 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TMThe analysis and separation of empty and full AAV9 capsid samples were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A) containing a complexing agent of 1 mM EDTA (or Na2-NTA, or Na3-citrate, or Na2-pyrophosphate), and further containing 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The majority of empty capsids and partially filled AAV capsids in the sample were eluted with a linear salt gradient of 40 CVs to the first elution salt (elution buffer, Buffer B): the elution buffer contained 60 mM sodium perchlorate, and further contained 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The flow rate was 1 mL / min. A second (stronger) elution salt (elution buffer, buffer C) was then used: the elution buffer contained 60 mM calcium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75. In this gradient, the vast majority of the fully capped AAV9 capsid was eluted. Finally, a high-salt wash (elution buffer, buffer D) was performed to remove any remaining binding material: the elution buffer contained 2000 mM sodium chloride, 1 mM Na2-EDTA (or Na2-NTA, or Na3-citrate, or Na2-pyrophosphate), 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75.

[0111] Results of various complexing agents such as Figure 6 As shown. Compared to Na2-NTA, complexing agents such as Na2-pyrophosphate, Na2-EDTA, or Na3-citrate are better able to separate fully capped caps from other caps. When using Na2-NTA, most empty caps, partially filled caps, and damaged caps are co-eluted with fully capped caps, resulting in a higher percentage of fully capped caps, at 79% and 76%, respectively.

[0112] Example 8: Effect of disodium complexing agents and fully deprotonated complexing agents on AAV9 capsid separation (1 mM Na2-EDTA vs. 1 mM Na4-EDTA) For 1 mM Na4-EDTA, use the following buffer: Buffer A: 20 mM BTP + 50 mM arginine + 1 mM Na4-EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 60 mM NaClO4 + 1% sorbitol + 0.01% poloxamer 188; pH 9.75 Buffer C: 20 mM BTP + 50 mM arginine + 60 mM CaCl2 + 1% sorbitol + 0.01% poloxamer 188; pH 9.75 Buffer D: 20 mM BTP + 50 mM arginine + 1 mM Na⁴-EDTA + 2000 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 CIMac column with 100 µL strong anion exchanger TM The analysis and separation of empty and full AAV9 capsid samples were performed on an AAV filled / empty column (Sardolibia Separation Company, Aidovschina, Slovenia). The column was equilibrated using loading conditions (loading and equilibration buffer, Buffer A) containing 1 mM Na4-EDTA complexing agent, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The majority of empty capsids and partially filled AAV capsids in the sample were eluted with a linear salt gradient of 40 CVs to the first elution salt (elution buffer, Buffer B): the elution buffer contained 65 mM sodium perchlorate, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol at pH 9.75. The flow rate was 1 mL / min. A second (stronger) elution salt (elution buffer, buffer C) was then used: the elution buffer contained 60 mM calcium chloride, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75. In this gradient, the vast majority of the fully capped AAV9 capsid was eluted. Finally, a high-salt wash (elution buffer, buffer D) was performed to remove any remaining binding material: the elution buffer contained 2000 mM sodium chloride, 1 mM Na4-EDTA, 50 mM arginine, 2.5% ethanol, 20 mM BTP, and 1% sorbitol, pH 9.75.

[0113] A comparison of the effects of disodium-type complexing agents and fully deprotonated complexing agents on AAV9 capsid separation, as follows: Figure 7 As shown. Figure 7The results showed that fully deprotonated EDTA (Na4-EDTA) only slightly altered the percentage of fully filled AAV9 capsids (from 43% to 41%), as well as the elution spectra of empty and / or partially filled AAV9 capsids. When using fully deprotonated EDTA, three peaks corresponding to empty and / or partially filled AAV9 capsids were observed. When using Na2-EDTA, the third peak in the second empty capsid peak was not observed.

[0114] Example 9: Analysis of purified AAV9 standard using PATfix AAV Switcher (Sardolibia Separation Company, Aidovschina, Slovenia) The buffer solution used for AEX separation is as follows: Buffer A: 20 mM BTP + 50 mM arginine + 0.5 mM Na2EDTA + 1% sorbitol + 2.5% EtOH; pH 9.75 Buffer B: 20 ​​mM BTP + 50 mM arginine + 60 mM NaClO4 + 1% sorbitol + 0.01% poloxamer; pH 9.75 Buffer C: 20 mM BTP + 50 mM arginine + 60 mM CaCl2 + 1% sorbitol + 0.01% poloxamer; pH 9.75 Buffer D: 20 mM BTP + 50 mM arginine + 0.5 mM Na2-EDTA + 2000 mM NaCl + 1% sorbitol + 2.5% EtOH; pH 9.75 On CIMac TM On an Adeno column, the separation of empty, partially filled, and fully capped capsids in complex lysate samples was achieved using a decreasing complexing agent gradient and a binary anion salt exchange gradient consisting of sodium perchlorate and calcium chloride. The lysate samples were first purified on a cation exchange column using a pH gradient (from pH 4.00 to pH 9.50). The pH gradient-eluted fractions were then transferred to a second AEX column, where the separation of multiple subpopulations was achieved. In addition to empty and fully capped capsids, partially filled capsids and other impurities were observed. Figure 8 ).

[0115] Example 10: Removal of complexing agent in sample and loading buffer enhances AAV9 capsid separation. The buffer solution used for AEX separation is as follows: Buffer A: 20 mM BTP + 1% sorbitol + 0.2% poloxamer 188; pH 9.30 (loading buffer) Buffer B: 20 ​​mM BTP + 1 mM trisodium citrate + 1% sorbitol + 0.2% poloxamer 188; pH 9.30 (wash buffer containing complexing agent) Buffer C: 20 mM BTP + 30 mM sodium perchlorate + 1% sorbitol + 0.2% poloxamer 188; pH 9.30 Buffer D: 20 mM BTP + 30 mM sodium perchlorate + 1% sorbitol + 0.2% poloxamer 188; pH 6.30 Buffer E: 0.5 M Tris + 1 M NaCl; pH ~9 (high-salt wash buffer) like Figure 9 As shown, in CIMac TM On an Adeno column, the separation of empty, partially filled, and fully filled AAV9 samples purified by DGUC was achieved. First, samples without complexing agents were loaded and then washed with a complexing agent. Most of the partially filled AAV9 capsids were eluted using an increasing salt gradient and a decreasing complexing agent gradient of 30 mM sodium perchlorate. By evaluating the UV ratio of the separation peaks [8], UV 260 / 280 ratios (1.17 and 1.19) indicated that these were partially filled AAV capsids. Most of the fully filled AAV9 capsids were eluted in a pH gradient, during which two distinct subpopulations of potentially fully filled AAV9 were observed with UV 260 / 280 ratios of 1.31 and 1.33, respectively [8]. Furthermore, a significant shift in retention time was observed in the fully filled AAV9 samples compared to those containing more partially filled capsids. This indicates that the separation is based on the length of the inserted fragment, in which the full capsid (due to its stronger electronegativity) will be eluted later in the gradient. With this modified method, empty AAV9 capsids were eluted in high-salt washes (with a UV 260 / 280 ratio of 0.62) [8]. This phenomenon suggests that when applied in the later stages of the method, the complexing agent may alter the elution spectrum of the empty capsids. Thus, this alteration may force them to elute towards the greater negative charge under high-salt gradients. Table 2: Orthogonal analysis mass spectrometry (AAV capsid percentage). Abbreviation Table AAV9 adeno-associated virus serotype 9 BTP (Bistripropane) Citrate CV column volume dPCR (Digital Polymerase Chain Reaction) EDTA (ethylenediaminetetraacetic acid) EtOH (ethanol) GFP (Green Fluorescent Protein) Kac potassium acetate LOD detection limit LOQ (Limit of Quantification) LNP lipid nanoparticles NTA (N-triacetic acid) PBS phosphate buffer PP pyrophosphate rAAV9 recombinant AAV9 vector genome with type 8 capsid Rep2-Cap8 plasmid expressing the Rep / Cap gene TFF tangential flow filter vg vector genome References [1] USP 621 Chromatography, December 2022.

[0116] [2] Yang Z (2009), “Hofmeister effect: an explanation of the influence of ionic liquids on biocatalysis”. Journal of Biotechnology. 144 (1): 12-22. doi:10.1016 / j.jbiotec.2009.04.011. PMID 19409939.

[0117] [3] Lock M, Vandenberghe LH, Wilson JM, Scalable Production Approach for AAV (US20160040137A1), 2016.

[0118] [4] P. Gagnon, B. Goricar, SD Prebil, H. Jug, M. Leskovec, A. Strancar, Separation of empty and intact adeno-associated virus capsids from weak anion exchangers by elution with an increasing pH gradient at low ionic strength, 2021.

[0119] [5] Yang Yun; Geng Xindu (2011), “Mixed-mode chromatography and its application in biopolymers.” Chromatography A. 1218 (49): 8813-8825. doi:10.1016 / j.chroma.2011.10.009. ISSN0021-9673. PMID 22033107.

[0120] [6] Zhao Guofeng; Dong Xiao-Yan; Sun Yan (2009), “Ligands for mixed-mode protein chromatography: principles, characteristics and design.” Journal of Biotechnology. 144(1): 3-11. doi:10.1016 / j.jbiotec.2009.04.009. ISSN 0168-1656. PMID 19409941.

[0121] [7] McLaughlin, Larry W. (1989). “Mixed-mode chromatography of nucleic acids.” Chemical Review. 89(2): 309-319. doi:10.1021 / cr00092a003. ISSN 0009-2665.

[0122] [8] Werle AK, Powers TW, Zobel JF, Waplehorst CN, Jarrold MF, Lyktey NA et al., (2021), “Comparison of analytical techniques for quantifying adeno-associated virus vector capsid content.” Molecular Therapy and Clinical Advances. 2021;23.

Claims

1. A method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material, the method comprising the following steps: - Load the ion exchange material with a loading buffer containing the drug delivery carrier mixture and a complexing agent, then... - Rinse the ion exchange material with an elution buffer containing a lower concentration of complexing agent than that in the loading buffer, or without a complexing agent. - Thus, the drug delivery carrier is eluted from the ion exchange material using the elution buffer, and optionally... - Collect the separated drug delivery carrier.

2. A method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material, the method comprising the following steps: - Load the ion exchange material with a loading buffer containing the drug delivery carrier mixture and free of complexing agents, then... - Rinse the ion exchange material with a washing buffer containing a complexing agent. - The drug delivery carrier is eluted from the ion exchange material using an elution buffer containing a lower concentration of a complexing agent than the washing buffer, or without a complexing agent, and optionally... - Collect the separated drug delivery carrier.

3. A method for chromatographic separation of a drug delivery carrier mixture in a buffered aqueous mixture using an ion exchange material, the method comprising the following steps: - Load the ion exchange material with a loading buffer containing the drug delivery carrier mixture, then - Rinse the ion exchange material with washing buffer. The loading buffer or the washing buffer, or both, contain a complexing agent. - The drug delivery carrier is eluted from the ion exchange material using an elution buffer, employing a salt gradient or pH gradient, wherein the elution buffer contains a lower concentration of a complexing agent than the loading buffer or the elution buffer, or contains no complexing agent, and optionally... - Collect the separated drug delivery carrier.

4. The method of any one of claims 1 to 3, wherein the delivery vector is selected from the group consisting of viruses, virus-like particles, extracellular vesicles, lipid nanoparticles or combinations thereof, preferably wherein the virus is parvovirus, AAV capsid, lentiviral capsid, adenovirus, or the extracellular vesicle is an exosome or bacteriophage; or a combination thereof.

5. The method of claim 4, wherein the virus is: (i) parvovirus, (ii) Adeno-associated viruses, particularly AAVs selected from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh.10, AAV11, AAV12, (iii) Mixed serotypes, especially recombinant mixed serotypes such as AAV2 / 8, (iv) AAV chimera, (v) Surface-modified AAV, or (vi) Synthetic derivative AAV-like particles.

6. The method of any one of claims 1 to 5, wherein the pharmaceutical preparation delivered by the delivery carrier is selected from the group consisting of genetic material such as DNA, RNA, or proteinaceous material such as antibodies, and non-biological material.

7. The method of any one of claims 1 to 6, wherein the ion exchange material is an anion exchanger, a cation exchanger, or a combination thereof, or wherein the ion exchange material is: (i) Mixed-mode chromatographic materials, (ii) Monolithic column anion or cation exchanger. (iii) Multi-mode materials for integral columns, (iv) Particulate anion or cation exchangers, and / or (v) Multimodal materials.

8. The method of any one of claims 1 to 7, wherein the ion exchange material device is used in: In the membrane, In granular form within a column, and / or In the fiber column.

9. The method of any one of claims 1 to 8, wherein the complexing agent is an organic compound.

10. The method of any one of claims 1 to 9, wherein the complexing agent is at least divalent, preferably wherein (i) the complexing agent comprises at least three chemical groups capable of providing a negative charge in an aqueous medium, such as carboxyl groups; or (ii) the complexing agent comprises at least three chemical groups capable of providing a positive charge in an aqueous medium, such as protonated amine groups.

11. The method according to any one of claims 1 to 10, wherein the complexing agent is selected from the group consisting of ethylenediaminetetraacetic acid, citric acid, pyrophosphate, diethyltriaminepentaacetic acid, tetrasodium glutamate diacetate, ethylene glycol-bis(oxyethyleneimino)tetraacetic acid, cyclohexanediaminetetraacetic acid and combinations thereof, or a polyamine salt, preferably selected from the group consisting of spermine, spermidine and combinations thereof.

12. The method of any one of claims 1 to 11, wherein the elution buffer comprises the following gradients: (i) an increasing salt concentration gradient, (ii) a pH gradient, or (iii) a combination of an increasing salt concentration gradient and a pH gradient.

13. The method of any one of claims 1 to 12, wherein the elution buffer initially contains a salt with low elution properties, which is replaced by a salt with high elution properties during elution.

14. The method of any one of claims 1 to 13, wherein the elution buffer comprises at least one stabilizer, preferably, wherein the stabilizer is selected from the group consisting of amino acids (particularly arginine, lysine, histidine), choline, glycopeptides, nucleotides, sugars, and combinations thereof.

15. The method of any one of claims 1 to 14, wherein the pH of the elution buffer is 3 to 10, or 7 to 10, particularly 9 to 10, or 9.

75.

16. The method of any one of claims 1 to 15, wherein the concentration gradient of the complexing agent in the elution buffer is reduced from 2 mM to 0.25 mM, particularly from 1.5 mM to 0.5 mM.

17. The method of any one of claims 1 to 16, wherein the elution buffer comprises at least one elution salt, at least one buffer, at least one isotonic substance, at least one auxiliary agent and / or at least one organic modifier.

18. The method of claim 17, wherein: - The isotonic substance is sucrose, sorbitol, mannitol, xylitol, or a mixture thereof; and / or - The auxiliary agent is a nonionic surfactant, such as poloxamer 188 or Tween. ® 20. Tween ® 80. Tergitol ® Triton X-100 or mixtures thereof; and / or - The organic modifier is acetonitrile, 1-butanol, tert-butanol, propylene carbonate, isopropanol, ethanol, methanol, ethylene glycol, 1-propanol or a mixture thereof.

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