Adeno-associated virus purification method

Abstract

Provided herein are methods for producing adeno-associated virus (AAV) products and methods for purifying adeno-associated virus. AAV is loaded onto an affinity resin, a wash step is performed at room temperature, and AAV is eluted from the affinity resin at a lower temperature. Various buffers for the wash step and elution are disclosed herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 868,282, filed on June 28, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to materials and methods for purifying adeno-associated viruses (AAV). Background Art

[0004] Adeno-associated virus (AAV) is a small non-enveloped virus that contains a linear single-stranded DNA genome. Since AAV's multiplicative infection occurs only in the presence of a helper virus (e.g., adenovirus or herpes virus), AAV belongs to the genus Dependovirus in the family Parvoviridae.

[0005] In order to safely use AAV in the clinic, several positions within the AAV genome have been genetically modified. For example, the Rep gene required for viral replication and elements required for site-specific integration have been removed from the AAV genome in many viral vectors. This recombinant AAV (rAAV) exists in an extrachromosomal state and has very low integration efficiency in genomic DNA. Therefore, the possibility of rAAV inducing random mutations in host cells is reduced, if not completely eliminated. Due to these characteristics and lack of pathogenicity, rAAV shows great promise as a gene therapy vector in many aspects of preclinical and clinical applications.

[0006] Although great efforts have been invested in designing efficient, large-scale methods to purify AAV products suitable for human administration, there is still a need for better AAV purification methods. During the purification of AAV, various other proteins and materials in the host cell culture matrix can be more effectively removed. Therefore, there is a need for AAV purification methods that include a step of removing host cell material from the final AAV product. Summary of the invention

[0007] A characteristic of producing AAV vectors in cell culture is the formation of a complex matrix comprising material from disrupted cells. In particular, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in the material from disrupted cells. The disclosed method includes a step of removing host cell material from the final AAV product under conditions that produce higher purity at a physiologically suitable pH.

[0008] In one aspect, provided herein is a method for purifying an adeno-associated virus (AAV), the method comprising:

[0009] (a) loading a solution containing AAV onto an affinity resin targeting an epitope on AAV under conditions that allow AAV in the solution to bind to the affinity resin;

[0010] (b) performing at least one washing step at room temperature; and

[0011] (c) AAV was eluted from the affinity resin at a temperature below 18°C.

[0012] The room temperature is 18° C. to 26° C. The room temperature may be 18° C., 18.5° C., 19° C., 19.5° C., 20° C., 20.5° C., 21° C., 21.5° C., 22° C., 22.5° C., 23° C., 23.5° C., 24° C., 24.5° C., 25° C., 25.5° C., or 26° C. The room temperature may be 18° C., about 18.5° C., about 19° C., about 19.5° C., about 20° C., about 20.5° C., about 21° C., about 21.5° C., about 22° C., about 22.5° C., about 23° C., about 23.5° C., about 24° C., about 24.5° C., about 25° C., about 25.5° C., or 26° C.

[0013] In some embodiments, the temperature in step (c) is 1° C. to 12° C. In some embodiments, the temperature in step (c) is 2° C. to 8° C. In some embodiments, the temperature in step (c) is 1° C., 1.5° C., 2° C., 2.5° C., 3° ​​C., 3.5° C., 4° C., 4.5° C., 5° C., 5.5° C., 6° C., 6.5° C., 7° C., 7.5° C., 8° C., 8.5° C., 9° C., 9.5° C., 10° C., 10.5° C., 11° C., 11.5° C., or 12° C. In some embodiments, the temperature in step (c) is about 1°C, about 1.5°C, about 2°C, about 2.5°C, about 3°C, about 3.5°C, about 4°C, about 4.5°C, about 5°C, about 5.5°C, about 6°C, about 6.5°C, about 7°C, about 7.5°C, about 8°C, about 8.5°C, about 9°C, about 9.5°C, about 10°C, about 10.5°C, about 11°C, about 11.5°C, or about 12°C.

[0014] In some embodiments, the method further comprises contacting the AAV-containing solution with an anion exchanger prior to loading the AAV-containing solution onto the affinity resin, and eluting the AAV-containing solution from the anion exchanger.

[0015] In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤ 99.9%. In some embodiments, the HC impurity level of the AAV obtained from the elution step is ≤ 99.0%.

[0016] In some embodiments, the AAV is AAV9. In some embodiments, the AAV9 comprises wild-type VP1. In some embodiments, the AAV9 comprises the VP1 of SEQ ID NO: 1.

[0017] In some embodiments, the method further comprises contacting the AAV-containing solution with a filter comprising positively charged groups effective to deplete the AAV-containing solution of acidic charged contaminants.

[0018] In some embodiments, the method further comprises: nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

[0019] In some embodiments, the method further comprises a polishing step comprising performing cation exchange chromatography.

[0020] In some embodiments, the method further comprises: detecting the AAV fraction by AAV-specific ELISA.

[0021] In some embodiments, the AAV-specific ELISA is a sandwich ELISA specific for AAV.

[0022] In another aspect, provided herein is an AAV product produced by any of the methods described above.

[0023] In one aspect, the present invention provides a method for purifying adeno-associated virus (AAV), which comprises: (a) loading a solution containing AAV onto an affinity resin for AAV at room temperature and under conditions that allow the AAV in the solution to bind to the affinity resin; (b) performing at least one washing step at room temperature; and (c) eluting AAV from the affinity resin at a temperature below 18°C.

[0024] In some embodiments, the temperature in step (c) is 1° C. to 12° C. In some embodiments, the temperature in step (c) is 2° C. to 8° C.

[0025] In some embodiments, the method further comprises contacting the AAV-containing solution with an anion exchanger prior to loading the AAV-containing solution onto the affinity resin, and eluting the AAV-containing solution from the anion exchanger.

[0026] In some embodiments, at least 2 washing steps are performed at room temperature. In some embodiments, at least 3 washing steps are performed at room temperature. In some embodiments, at least 4 washing steps are performed at room temperature.

[0027] In some embodiments, 2 washing steps are performed. In some embodiments, 3 washing steps are performed. In some embodiments, 4 washing steps are performed.

[0028] In some embodiments, the washing steps are performed continuously.

[0029] In some embodiments, at least one wash buffer comprises about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, at least one wash buffer comprises about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In some embodiments, at least one wash buffer comprises about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, at least one wash buffer comprises about 50mM TrisHCl and about 125mM salt. In some embodiments, the pH of the wash buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5. In some embodiments, at least one wash buffer comprises about 50mMTrisHCl and about 125mM salt, and the pH is about 8.5.

[0030] In some embodiments, at least one wash buffer comprises about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6.5, or about 6.0. In some embodiments, at least one wash buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0031] In some embodiments, at least one wash buffer comprises about 10mM to about 200mM TrisHCl and about 10% to about 75% (w / w) ethylene glycol. In some embodiments, at least one wash buffer comprises about 25mM to about 100mM TrisHCl and about 25% to about 70% (w / w) ethylene glycol. In some embodiments, at least one wash buffer comprises about 40mM to about 60mM TrisHCl and about 40% to about 60% (w / w) ethylene glycol. In some embodiments, at least one wash buffer comprises about 50mM TrisHCl and about 50% (w / w) ethylene glycol. In some embodiments, the pH of the wash buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5. In some embodiments, at least one wash buffer comprises about 50mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH is about 8.5.

[0032] In some embodiments, at least one wash buffer comprises about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% (w / w) trehalose, and about 0.0005% to about 1% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% (w / w) trehalose, and about 0.001% to about 0.1% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% (w / w) trehalose, and about 0.001% to about 0.05% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose, and about 0.005% (w / w) polysorbate 80. In some embodiments, the pH of the wash buffer is about 6.0 to about 8.0, about 6.5 to about 7.5, about 7.0 to about 7.4, or about 7.0. In some embodiments, at least one wash buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose, and about 0.005% (w / w) polysorbate 80 at a pH of about 7.0.

[0033] In some embodiments, at least one wash buffer comprises about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, at least one wash buffer comprises about 20 mM TrisHCl, about 150 mM salt, and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the wash buffer is about 6.0 to about 8.8, about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.4. In some embodiments, at least one wash buffer comprises about 20 mM TrisHCl, about 150 mM salt, and about 0.1% (w / w) polysorbate 80 at a pH of about 7.4.

[0034] In some embodiments, step (c) comprises eluting the AAV with at least one elution buffer. In some embodiments, at least one elution buffer is the same as at least one wash buffer. In some embodiments, at least one elution buffer is the same as the last wash buffer used in the last wash step before eluting the AAV in step (c). In some embodiments, the first elution buffer is the same as the last wash buffer used in the last wash step before eluting the AAV in step (c).

[0035] In some embodiments, at least one elution buffer comprises about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, at least one elution buffer comprises about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In some embodiments, at least one elution buffer comprises about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, at least one elution buffer comprises about 50mM TrisHCl and about 125mM salt. In some embodiments, the pH of the elution buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5. In some embodiments, at least one elution buffer comprises about 50mMTrisHCl and about 125mM salt, and the pH is about 8.5.

[0036] In some embodiments, at least one elution buffer comprises about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the elution buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6.5, or about 6.0. In some embodiments, at least one elution buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0037] In some embodiments, at least one elution buffer comprises about 10mM to about 200mM TrisHCl and about 10% to about 75% (w / w) ethylene glycol. In some embodiments, at least one elution buffer comprises about 25mM to about 100mM TrisHCl and about 25% to about 70% (w / w) ethylene glycol. In some embodiments, at least one elution buffer comprises about 40mM to about 60mM TrisHCl and about 40% to about 60% (w / w) ethylene glycol. In some embodiments, at least one elution buffer comprises about 50mM TrisHCl and about 50% (w / w) ethylene glycol. In some embodiments, the pH of the elution buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5. In some embodiments, at least one elution buffer comprises about 50mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH is about 8.5.

[0038] In some embodiments, at least one elution buffer comprises about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% (w / w) trehalose, and about 0.0005% to about 1% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% trehalose, and about 0.001% to about 0.1% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% (w / w) trehalose, and about 0.001% to about 0.05% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose, and about 0.005% (w / w) polysorbate 80. In some embodiments, the pH of the elution buffer is about 6.0 to about 8.0, about 6.5 to about 7.5, about 7.0 to about 7.4, or about 7.0. In some embodiments, at least one elution buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose, and about 0.005% (w / w) polysorbate 80 at a pH of about 7.0.

[0039] In some embodiments, at least one elution buffer comprises about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, at least one elution buffer comprises about 20 mM TrisHCl, about 150 mM salt, and 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the elution buffer is about 6.0 to about 8.8, about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.4. In some embodiments, at least one elution buffer comprises about 20 mM TrisHCl, about 150 mM salt, and 0.1% (w / w) polysorbate 80, and has a pH of about 7.4.

[0040] In some embodiments, the first washing step, the second washing step, the third washing step and / or the fourth washing step include adding a buffer to the affinity resin, the buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the first washing step, the second washing step, the third washing step and / or the fourth washing step include adding a buffer to the affinity resin, the buffer comprising about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In some embodiments, the first washing step, the second washing step, the third washing step and / or the fourth washing step include adding a buffer to the affinity resin, the buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the first washing step, the second washing step, the third washing step and / or the fourth washing step include adding a buffer to the affinity resin, the buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprise adding a buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprise adding a buffer having a pH of about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0041] In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprise adding a buffer to the affinity resin, the buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprise adding a buffer to the affinity resin, the buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprise adding a buffer to the affinity resin, the buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprises adding a buffer to the affinity resin, the buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the first wash step, the second wash step, the third wash step, and / or the fourth wash step is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the first wash step, the second wash step, the third wash step, and / or the fourth wash step comprises adding a buffer to the affinity resin, the buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH is about 6.0.

[0042] In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the first washing step includes adding a first buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the first wash step comprises adding a first buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0043] In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the second wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the second wash step comprises adding a second buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0044] In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the third washing step includes adding a third buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the third wash step comprises adding a third buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0045] In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 40mM to about 60mMTrisHCl and about 100mM to about 150mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the elution washing step includes adding a buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, step (c) comprises adding a buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0046] In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the first washing step includes adding a first buffer to the affinity resin, the first buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the first washing step includes adding a first buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the first wash step comprises adding a first buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0047] In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the second wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the second wash step comprises adding a second buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0048] In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the third washing step includes adding a third buffer to the affinity resin, the third buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the third washing step includes adding a third buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the third wash step comprises adding a third buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0049] In some embodiments, the fourth wash step comprises adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the fourth wash step comprises adding a fourth buffer to the affinity resin, the fourth buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the fourth wash step comprises adding a fourth buffer to the affinity resin, the fourth buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the fourth wash step comprises adding a fourth buffer to the affinity resin, the fourth buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the fourth wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the fourth wash step comprises adding a fourth buffer to the affinity resin, the fourth buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0050] In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the elution buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, step (c) comprises adding a buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0051] In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the first wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the first wash step comprises adding a first buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0052] In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the second washing step includes adding a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the second wash step comprises adding a second buffer to the affinity resin, wherein the second buffer comprises about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5.

[0053] In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the third wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the third wash step comprises adding a third buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0054] In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, step (c) comprises adding a buffer to the affinity resin, the buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the elution buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, step (c) comprises adding a buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0055] In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the first wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the first wash step comprises adding a first buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0056] In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the second washing step includes adding a second buffer to the affinity resin, the second buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the second washing step includes adding a second buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the second wash step comprises adding a second buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0057] In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In some embodiments, the third wash step comprises adding a third buffer to the affinity resin, the third buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In some embodiments, the pH of the third buffering step is about 5.0 to about 7.4, about 5.5 to about 7.0, about 5.5 to about 6, or about 6. In some embodiments, the third wash step comprises adding a third buffer comprising about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0 to the affinity resin.

[0058] In some embodiments, the fourth washing step includes adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, the fourth washing step includes adding a fourth buffer to the affinity resin, the fourth buffer comprising about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt. In some embodiments, the fourth washing step includes adding a fourth buffer to the affinity resin, the fourth buffer comprising about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In some embodiments, the fourth washing step includes adding a fourth buffer to the affinity resin, the fourth buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the fourth washing step includes adding a fourth buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, the fourth wash step comprises adding a fourth buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0059] In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 40mM to about 60mMTrisHCl and about 100mM to about 150mM salt. In some embodiments, step (c) includes adding a buffer to the affinity resin, the buffer comprising about 50mM TrisHCl and about 125mM salt. In some embodiments, the elution washing step includes adding a buffer having a pH of about 7.5 to about 9.2, about 8.0 to about 9.0, about 8.0 to about 8.8, or about 8.5 to the affinity resin. In some embodiments, step (c) comprises adding a buffer comprising about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5 to the affinity resin.

[0060] In some embodiments (such as the above embodiments), the salt is selected from: NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments (such as the above embodiments), the salt is NaCl.

[0061] In some embodiments, the buffer comprises about 50 mM TrisHCl and about 125 mM NaCl at a pH of about 8.5.

[0062] In some embodiments, the purity level of the AAV obtained from the elution step is 99.9% or more. In some embodiments, the purity level of the AAV obtained from the elution step is 99.0% or more.

[0063] In some embodiments, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the AAV capsids eluted by elution step (c) are intact AAV capsids.

[0064] In some embodiments, the affinity resin is an AAVx resin.

[0065] In some embodiments, the AAV is AAV9. In some embodiments, the AAV9 comprises a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, and / or SEQ ID NO:3.

[0066] In some embodiments, the method further comprises contacting the AAV-containing solution with a filter comprising positively charged groups effective to deplete the AAV-containing solution of acidic charged contaminants.

[0067] In some embodiments, the method further comprises: nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

[0068] In some embodiments, the method further comprises a polishing step comprising performing cation exchange chromatography.

[0069] In some embodiments, the method further comprises: detecting the AAV fraction by an AAV-specific ELISA. In some embodiments, the AAV-specific ELISA is a sandwich ELISA specific for AAV.

[0070] In another aspect, provided herein is an AAV product produced by the method according to any one of the embodiments disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 A chromatogram of the separation procedure according to Example 3 is shown.

[0072] Figure 2A and Figure 2B A chromatogram is shown for the separation procedure according to Example 4. The application area and the wash-elution area are separated by the "split screen" function. Blue: UV 280 nm, purple: UV 254 nm, red: conductivity.

[0073] Figure 3 A chromatogram according to the separation procedure of Example 6 is shown. DETAILED DESCRIPTION

[0074] Provided herein are methods for producing adeno-associated virus (AAV) products, methods for purifying AAV, and methods for purifying intact AAV capsids from a concentrated AAV fraction comprising empty AAV capsids and intact AAV capsids.

[0075] A characteristic of AAV vector production in cell culture is the formation of a complex matrix comprising material from disrupted cells. In particular, host cell proteins, proteasomes, cell debris, and potential virus-specific receptors are often present in the material from disrupted cells. The disclosed method includes a step of removing host cell material from the final AAV product under conditions that produce higher purity at a physiologically suitable pH.

[0076] In one aspect, provided herein is a method for purifying an adeno-associated virus (AAV), the method comprising:

[0077] (a) loading a solution containing AAV onto an affinity resin targeting an epitope on AAV under conditions that allow AAV in the solution to bind to the affinity resin;

[0078] (b) performing at least one washing step at room temperature; and

[0079] (c) AAV was eluted from the affinity resin at a temperature below 18°C.

[0080] In some aspects, the method comprises eluting the AAV capsid from the affinity resin by lowering the buffer temperature from room temperature. In some embodiments, the AAV can be eluted from the same resin using the same buffer at a reduced temperature of 1°C to 12°C. In some embodiments, the AAV can be eluted from the same resin using the same buffer at a reduced temperature of 2°C to 8°C. Increasing the elution of AAV by lowering the temperature from room temperature is surprising and unexpected. Without being bound by a particular theory, this result is surprising and unexpected because, although lower temperatures are known to prolong the time for the binding between the antibody and the antigen to reach equilibrium, one skilled in the art would not expect that the already bound antigen would be readily released when the temperature is lowered to below room temperature.

[0081] The temperature change elution scheme has the benefit of gentle elution at low temperature to help maintain the structure and / or infectivity of the AAV particles. The elution according to various embodiments described herein can prevent exposure to low pH (e.g., elution at a pH close to neutral) and maintain high titer of AAV. In addition, the use of a mild elution buffer can be easy to implement in a manufacturing environment and more efficient because the elution step does not require replacement of the buffer. In addition, the temperature change elution scheme allows a higher content of complete AAV capsids.

[0082] In certain embodiments, AAV9 can be bound to a resin described herein at a temperature of about 20°C to 25°C and can be eluted at a lower temperature of about 1°C to 12°C in the same buffer system. In certain embodiments, AAV9 can be bound to a resin described herein at a temperature of about 20°C to 25°C and can be eluted at a lower temperature of about 2°C to 8°C in the same buffer system. For example, in a buffer comprising 125 mM NaCl and 50 mM TrisHCl at pH 8.5, AAV9 can be bound to CaptureSelect AAVx resin at room temperature and can be eluted from the resin using the same buffer at a lower temperature of about 1°C to 12°C or about 2°C to 8°C. For another example, in a buffer comprising 100 mM sodium acetate and 0.1% (w / w) polysorbate 80 at pH 6.0, AAV9 can be bound to CaptureSelect AAVx resin at room temperature and can be eluted from the resin using the same buffer at a lower temperature of about 1°C to 12°C or about 2°C to 8°C.

[0083] Further improvements in AAV product purity can be seen when the wash step and the elution step are performed sequentially according to the various embodiments and examples described herein.

[0084] definition

[0085] Unless otherwise indicated herein or clearly contradicted by context, the terms "a", "an", and "the" and similar referents used in the context of this disclosure (especially in the context of the following claims) should be construed to cover both the singular and the plural. Unless otherwise indicated, the terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise indicated.

[0086] As used herein, the terms "capsid," "capsid particle," and "particle" are used interchangeably to refer to an AAV particle composed of at least one complete AAV capsid.

[0087] As used herein, the term "empty (empty)" about AAV or AAV capsid refers to those lacking a complete (i.e., complete) target gene (GOI). Empty AAV or empty AAV capsid or empty AAV particles cannot provide therapeutic benefits. As used herein, the term "full (full)" about AAV or AAV capsid or AAV particles refers to those containing most of the complete GOI. The complete AAV capsid can provide therapeutic benefits for recipient patients. In certain embodiments, "complete" can also include "incomplete vector DNA" or "truncated vector DNA". In certain embodiments, "overfilled (overfilled)" about AAV or AAV capsid or AAV particles refers to those that may contain double-packaged or longer genomes or GOIDNA (e.g., up to twice the size). In certain embodiments, complete and incomplete and / or truncated and / or overfilled vector DNA can be distinguished by other analytical methods. Such methods include, but are not limited to, determining DNA size by capillary electrophoresis, AUC (analytical ultracentrifugation), % agarose DNA (native or alkaline), gel, Southern Blot, dot hybridization, UV spectrophotometry, weak anion exchange chromatography, and mass spectrometry (see Resolving Adeno-Associated Viral Particle Diversity with Charge Detection Mass Spectrometry, Elizabeth E. Pierson et al., Anal. Chem., 2016, 88(13), pp. 6718-6725, which is incorporated herein in its entirety for all purposes).

[0088] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0089] Unless otherwise specified herein or the context is clearly contradictory, all methods described herein can be performed in any suitable order. Unless otherwise stated, the use of any and all examples or exemplary language (e.g., "for example") provided herein is intended only to better illustrate the present disclosure and does not limit the scope of the present disclosure. No language in the specification should be interpreted as indicating that any unclaimed element is essential to the implementation of the present disclosure.

[0090] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventor for implementing the present disclosure. After reading the above description, variations of those preferred embodiments will be apparent to those of ordinary skill in the art. The inventors expect that those skilled in the art will appropriately use these variations, and the inventors intend to implement the present disclosure in a manner different from that specifically described herein. Therefore, the present disclosure includes all variations and equivalents of the subject matter described in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or the context is clearly contradictory, the present disclosure covers any combination of all possible variations of the above elements.

[0091] Washing steps, elution steps and buffers

[0092] In one aspect, the present invention provides a method for purifying adeno-associated virus (AAV). The method comprises: (a) loading a solution containing AAV onto an affinity resin for AAV under conditions that allow AAV in the solution to bind to the affinity resin; (b) performing at least one washing step at room temperature; (c) eluting AAV from the affinity resin at a temperature below 18°C.

[0093] The affinity purification step includes one or more washing steps. One or more washing steps may be followed by one or more elution steps. In certain embodiments, the methods of the present disclosure include a filtration step that occurs prior to the affinity purification step.

[0094] In some embodiments, at least 2 washing steps are performed, each involving the same or different buffer. In some embodiments, at least 3 washing steps are performed, each involving the same or different buffer. In some embodiments, at least 4 washing steps are performed, each involving the same or different buffer. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 washing steps are performed, each involving the same or different buffer. In some embodiments, 2 washing steps are performed. In some embodiments, 3 washing steps are performed. In some embodiments, 4 washing steps are performed. In certain embodiments, the washing buffers are different. In some embodiments, the washing steps are performed continuously. One or more of these washing steps or even all of these washing steps are performed at room temperature (e.g., 18°C ​​to 26°C, or 18°C, 18.5°C, 19°C, 19.5°C, 20°C, 20.5°C, 21°C, 21.5°C, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C, or 26°C). In certain embodiments, all washing steps are performed at room temperature.

[0095] In certain embodiments, at least one wash buffer is used. In certain embodiments, at least two different wash buffers can be used. In certain embodiments, at least three different wash buffers can be used. In certain embodiments, at least four different wash buffers can be used. In certain embodiments, one wash buffer can be used. In certain embodiments, two different wash buffers can be used. In certain embodiments, three different wash buffers can be used. In certain embodiments, four different wash buffers can be used.

[0096] In certain embodiments, at least one elution step is performed. In certain embodiments, at least 2 elution steps are performed, each involving the same or different buffer. In certain embodiments, at least 3 elution steps are performed, each involving the same or different buffer. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 elution steps are performed, each involving the same or different buffer. In certain embodiments, one elution step is performed. In certain embodiments, at least 2 elution steps are performed. In certain embodiments, at least 3 elution steps are performed. In certain embodiments, at least one elution buffer is the same as at least one wash buffer. In certain embodiments, at least one elution buffer is different from a wash buffer. In certain embodiments, at least one elution buffer is the same as the last wash buffer used in the last wash step before eluting the AAV. In certain embodiments, the first elution buffer is the same as the last wash buffer used in the last wash step before eluting the AAV. Elution is performed at a temperature of 1° C. to 12° C. (e.g., 2° C. to 8° C., or 1° C., 1.5° C., 2° C., 2.5° C., 3° ​​C., 3.5° C., 4° C., 4.5° C., 5° C., 5.5° C., 6° C., 6.5° C., 7° C., 7.5° C., 8° C., 8.5° C., 9° C., 9.5° C., 10° C., 10.5° C., 11° C., 11.5° C., or 12° C.). Elution according to various embodiments described herein can prevent exposure to low pH (e.g., elution at a pH close to neutral) and maintain high titer of AAV.

[0097] In certain embodiments, at least one elution buffer is used. In certain embodiments, at least two different elution buffers may be used. In certain embodiments, at least three different elution buffers may be used. In certain embodiments, at least four different elution buffers may be used. In certain embodiments, one elution buffer may be used. In certain embodiments, two different elution buffers may be used. In certain embodiments, three different elution buffers may be used. In certain embodiments, four different elution buffers may be used.

[0098] In certain embodiments, the temperature can be changed to below room temperature by a cooling cabinet (e.g., Unichromat 1500), a temperature jacket (e.g., a water cooling jacket), and / or the use of a cold buffer for elution. In certain embodiments, the buffer is prepared at room temperature before cooling. In certain embodiments, the pH of the buffer is measured at room temperature before cooling.

[0099] Various volumes can be used, such as about 2× column volumes to about 15× column volumes, about 3× column volumes to about 7× column volumes, about 4× column volumes to about 8× column volumes, about 5× column volumes to about 10× column volumes, or about 7× column volumes to about 12× column volumes. For example, when the column volume is about 2 mL to about 3 mL, 10× column volumes can be used. Any wash buffer and / or elution buffer of about 5× column volumes or 5× column volumes can be used. Alternatively, any wash buffer and / or elution buffer of about 10× column volumes or 10× column volumes can be used. The time of the wash step can be further extended to increase the purity of the AAV.

[0100] The wash steps can effectively remove strongly bound contaminants from the AAV and / or the base resin of the affinity matrix. At the same time, the buffer used in the wash steps will not substantially elute the AAV.

[0101] In certain embodiments, at least one wash buffer comprises a chelating agent (e.g., EDTA). In certain embodiments, the wash buffer comprises a single amino acid or any combination of two or more amino acids that ensures the pH range and depletion rate of the host cell (e.g., HEK-HCP), such as glycine, arginine, tryptophan, amino derivatives such as taurine (oxidized cysteine), N-acetyl-tryptophan, and glycylglycine.

[0102] The elution step can effectively elute the AAV capsid. In some embodiments, the elution step preferably elutes the complete AAV capsid relative to the empty AAV capsid or the overfilled AAV capsid. In some embodiments, the elution buffer comprises a single amino acid or any combination of two or more amino acids to ensure pH and elution of AAV, such as glycine, arginine, tryptophan, amino derivatives such as taurine (oxidized cysteine), N-acetyl-tryptophan and glycine.

[0103] Without wishing to be bound by theory, the extent of elution of AAV is affected by both the amount of ethylene glycol in the third buffer and the conductivity of the salt. An amount of at least 55% (w / w) ethylene glycol in the buffer can significantly increase the amount of elution compared to 50% (w / w) ethylene glycol. Thus, at a given ethylene glycol concentration, an increased NaCl concentration can increase the extent and rate of elution. At a given ethylene glycol concentration, replacing NaCl with a multivalent salt can also increase the extent and rate of elution.

[0104] Without wishing to be bound by theory, if the salt is constant (e.g., 150 mM NaCl), increasing the amount of ethylene glycol can increase the elution strength of the buffer. If the ethylene glycol content is constant (e.g., 55%), increasing the amount of salt can increase the elution strength of the buffer. Thus, in 150 mM NaCl, the elution strength increases from 40% to 45% to 50% to 55% to 60% (w / w) ethylene glycol. Increasing the ethylene glycol content of a solution with a constant salt content decreases conductivity. An increase in the amount of ethylene glycol decreases the solubility of the salt in the buffer.

[0105] In certain embodiments, one or more of sorbitol, mannitol, xylitol, sucrose, trehalose, glycerol (1,2,3-propanetriol) or erythritol (meso-1,2,3,4-butanediol) may be used in combination with ethylene glycol or in place of ethylene glycol. In certain embodiments, the elution buffer may include about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 48% to about 52%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, or about 70% to about 75% (w / w) ethylene glycol. In certain embodiments, the elution buffer may include about 50% or 50% (w / w) ethylene glycol. In certain embodiments, the concentration of ethylene glycol is at least 55% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 56% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 57% (w / w). In certain embodiments, the concentration of ethylene glycol is at least 58% (w / w).

[0106] For example, the wash buffer and / or elution buffer may comprise one or more of the following: TrisHCl, acetate, phosphate, histidine, imidazole, lysine, arginine, glycine, taurine, citrate, HEPES, MES, MES-Na, borate, Bis-Tris, MOPS, Bicine, Tricine, TAPS, TAPSO, MES, PIPES, TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), barbital sodium (Veronal), ADA (N-(2-acetamido)iminodiacetic acid), ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), Bis-Tris propane, BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), DIPSO ( 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid), Trizma, HEPPSO (4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid)), POPSO (piperazine-1,4-bis(2-hydroxypropanesulfonic acid) dehydrate), TEA, EPPS (4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPBS (N-(2-hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), AMPD (2-amino-2-methyl-1,3-propanediol), AMPSO (N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid), a single amino acid or any combination of two or more amino acids, such as glycine, arginine, tryptophan, amino derivatives such as taurine (oxidized cysteine), N-acetyl-tryptophan and glycylglycine.

[0107] In some embodiments, the wash buffer and / or elution buffer may contain sodium acetate, TrisHCl, arginine-HCl, lysine-HCl, histidine-HCl, histidine, glycine, taurine, MES-Na, Bis-Tris, citrate, acetate, MES, HEPES, phosphate, TrisHCl, Bis-Tris, histidine, imidazole, ArgininHCl, LysinHCl, glycine, glycylglycine, borate, MOPS, Bicine, Tricine, TAPS, TAPSO, PIPES, L-glutamic acid, aspartic acid, BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and / or N-acetyl-D,L-tryptophan.

[0108] In certain embodiments, the wash buffer and / or elution buffer further comprises salt. In some embodiments, the buffer comprises TrisHCl and salt. In some embodiments, the buffer comprises arginine-HCl and salt. In some embodiments, the buffer comprises histidine and salt.

[0109] For the wash buffer and / or elution buffer described herein, the salt can be selected from NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, and a combination of one or more of NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, C2H3NaO2, sodium citrate, and potassium citrate. In some embodiments, the salt concentration is about 50mM to about 2000mM, about 100mM to about 1500mM, about 100mM to about 200mM, about 200mM to about 1000mM, about 500mM to about 900mM, about 600mM to about 800mM, about 750mM, or 750mM. In some embodiments, the salt concentration is about 50mM to about 2000mM, about 100mM to about 1500mM, about 100mM to about 200mM, about 200mM to about 1000mM, about 500mM to about 900mM, about 600mM to about 800mM NaCl, about 750mM NaCl or 750mM NaCl. In some embodiments, when a concentration gradient of NaCl is used, the target concentration is 2000mM. In some embodiments, the concentration of the salt does not exceed 500mM. In some embodiments, the concentration of the salt does not exceed 200mM. In some embodiments, the salt is NaCl. In some embodiments, the salt is 125mM NaCl. In some embodiments, the salt is 150mM NaCl.

[0110] In certain embodiments, the washing buffer and / or the elution buffer may further comprise one or more organic solvents or detergents. For example, the organic solvent or detergent may be, but is not limited to, Tween 80, polysorbate 80, Triton X100, tri(n-butyl) phosphate (TNBP), ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose or trehalose. For example, the detergent may be, but is not limited to, polyoxyethylene nonionic surfactants (e.g., Brij 35), 4-nonylphenyl-polyethylene glycol (Arkopal N100), octyl glucoside, n-dodecyl β-D-maltoside, digitonin, 6-cyclohexylhexyl β-D-maltoside or octyl pyranoglucoside. For example, ethylene glycol may be PEG, such as, but not limited to, PEG 2000, PEG 4000, PEG 6000 (Macrogol). For example, the organic solvent may be, but is not limited to, glycerol (1,2,3-propanetriol) and erythritol (meso-1,2,3,4-butanediol). In some embodiments, the detergent comprises one or more of Triton X100, polysorbate 80, and tri(n-butyl) phosphate (TNBP). In some embodiments, the organic solvent or detergent may be polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, sucrose, or trehalose. In some embodiments, the buffer comprises TrisHCl and DMSO.

[0111] In certain embodiments, the wash buffer and / or elution buffer comprises about 0.0005% to about 20%, about 0.0005% to about 15%, about 0.0005% to about 10%, about 0.0005% to about 5%, about 0.0005% to about 1%, about 0.001% to about 4%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.005% to about 3%, about 0.01% to about 2.5%, about 0.05% to about 5%, about 0.05% to about 2%, 0.05% to about 0.2%, or about 0.1% to about 1.5% (w / w) of an organic solvent or detergent. In some embodiments, the organic solvent or detergent is present at about 0.005% (w / w). In some embodiments, the organic solvent or detergent is present at about 0.1% (w / w).

[0112] In some embodiments, the organic solvent or detergent is polysorbate 80 (e.g., Tween 80 or Crillet). In some embodiments, the buffer comprises polysorbate 80. In some embodiments, the buffer comprises arginine-HCl and polysorbate 80. In some embodiments, the buffer comprises taurine and polysorbate 80. In some embodiments, the buffer comprises TrisHCl and polysorbate 80. In some embodiments, the buffer comprises sodium acetate and polysorbate 80.

[0113] In certain embodiments, the wash buffer and / or elution buffer comprises about 0.0005% to about 20%, about 0.0005% to about 15%, about 0.0005% to about 10%, about 0.0005% to about 5%, about 0.0005% to about 1%, about 0.001% to about 4%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.005% to about 3%, about 0.01% to about 2.5%, about 0.05% to about 5%, about 0.05% to about 2%, 0.05% to about 0.2%, or about 0.1% to about 1.5% (w / w) polysorbate 80. In some embodiments, the wash buffer and / or elution buffer comprises about 0.005% (w / w) polysorbate 80. In some embodiments, the wash buffer and / or elution buffer comprises about 0.1% (w / w) polysorbate 80. In some embodiments, the wash buffer and / or elution buffer comprises about 5% (w / w) polysorbate 80. In some embodiments, the wash buffer and / or elution buffer comprises about 10% (w / w) polysorbate 80. In some embodiments, the wash buffer and / or elution buffer comprises about 20% (w / w) polysorbate 80.

[0114] In certain embodiments, the wash buffer and / or elution buffer may comprise about 30% to about 35%, 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 48% to about 52%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, or about 70% to about 75% (w / w) ethylene glycol. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50% or 50% (w / w) ethylene glycol.

[0115] The organic solvent or detergent need not be present in all wash buffers and / or elution buffers used. In certain embodiments, the organic solvent or detergent is not present in any wash buffer and / or elution buffer used. In certain embodiments, the organic solvent or detergent is present in at least one wash buffer used. In certain embodiments, the organic solvent or detergent is present in at least one elution buffer used. In some embodiments, the wash buffer (e.g., the first wash buffer) comprises sodium acetate and polysorbate 80. In some embodiments, the wash buffer (e.g., the second wash buffer) comprises sodium acetate and polysorbate 80. In some embodiments, the wash buffer comprises one or more of Tween 80, DMSO and tri(n-butyl) phosphate (TNBP). In some embodiments, the wash buffer comprises one or more of Triton-X 100, polysorbate 80 and TNBP. In some embodiments, the wash buffer (e.g., the third wash buffer) comprises Tris and ethylene glycol. Without wishing to be bound by theory, the organic solvents and detergents in the wash buffer may effectively remove strongly bound host proteins and viral receptors while also inactivating and / or degrading lipid enveloped viruses.

[0116] In some embodiments, the buffer further comprises ethylene glycol, sucrose, taurine and / or glycerol. In some embodiments, the buffer comprises one of arginine-HCl and sucrose and glycerol. In some embodiments, the buffer comprises taurine and ethylene glycol. In some embodiments, the buffer comprises TrisHCl and ethylene glycol. In some embodiments, the buffer comprises sodium acetate and ethylene glycol.

[0117] In certain embodiments, wash buffer and / or elution buffer can be a buffer based on Tris comprising salt (e.g., NaCl). In certain embodiments, wash buffer and / or elution buffer can be a buffer based on sodium acetate (NaAc). In certain embodiments, wash buffer and / or elution buffer can include sodium salt (MES-Na) of 2-(N-morpholino) ethanesulfonic acid, EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, wash buffer and / or elution buffer can include about 50mM to about 200mM taurine and 0.2% to 1.5% PEG (e.g., PEG 6000). In certain embodiments, wash buffer and / or elution buffer can include Bis-Tris, and a solvent / detergent mixture containing Triton-X 100, polysorbate 80, and TNBP. In certain embodiments, wash buffer and / or elution buffer may include: a glycine-based buffer, a sodium citrate-based buffer, or an arginine-HCl-based buffer comprising a salt (e.g., NaCl). In certain embodiments, wash buffer and / or elution buffer may be a Tris-based buffer, a taurine-based buffer, or an arginine-HCl-based buffer comprising ethylene glycol and / or NaCl. Alternatively, one or more of sorbitol, mannitol, xylitol, sucrose, or trehalose may be used in combination with ethylene glycol or replace ethylene glycol. In certain embodiments, wash buffer and / or elution buffer may include sodium acetate and polysorbate 80.

[0118] In certain embodiments, wash buffer and / or elution buffer comprises about 10mM to about 500mM TrisHCl. In certain embodiments, wash buffer and / or elution buffer comprises about 10mM to about 400mM, about 10mM to about 300mM, about 10mM to about 200mM, about 15mM to about 175mM, about 20mM to about 150mM, about 25mM to about 125mM, about 25mM to about 100mM, about 30mM to about 90mM, about 35mM to about 75mM, or about 40mM to about 60mM TrisHCl. In certain embodiments, the wash buffer and / or elution buffer comprises about 10 mM to about 15 mM, about 10 mM to about 30 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 30 mM to about 35 mM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 40 mM to about 50 mM, about 45 mM to about 50 mM, about 50 mM to about 55 mM, about 55 mM to about 60 mM, about 60 mM to about 65 mM, about 65 mM to about 70 mM. In some embodiments, the wash buffer and / or elution buffer may comprise about 50mM or 50mM TrisHCl. In some embodiments, the wash buffer and / or elution buffer may comprise about 20mM or 20mM TrisHCl.

[0119] In certain embodiments, the wash buffer and / or elution buffer may comprise about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about about 500mM, about 525mM, about 550mM, about 575mM, about 600mM, about 625mM, about 650mM, about 675mM, about 700mM, about 725mM, about 750mM, about 775mM, about 800mM, about 825mM, about 850mM, about 875mM, about 900mM, about 925mM, about 950mM, about 975mM, about 1000mM, about 1025mM, about 1050mM, about 1075mM, about 1100mM, about 1125mM, about 1150mM, about 1175mM, about 1200mM, about 1225mM, about 1250mM, about 1275mM, about 1300mM, about 1325mM, about 1350mM, about 1375mM, about 1400mM, about 1425mM, about 1450mM, about 1475mM, about 1500mM, about 15 In some embodiments, wash buffer and / or elution buffer may comprise about 50mM or 50mM TrisHCl. In some embodiments, wash buffer and / or elution buffer may comprise about 20mM or 20mM TrisHCl.

[0120] In certain embodiments, TrisHCl wash buffer and / or elution buffer may also include a salt of about 50mM to about 500mM. In certain embodiments, TrisHCl wash buffer and / or elution buffer may also include a salt of about 55mM to about 400mM, about 60mM to about 350mM, about 70mM to about 300mM, about 75mM to about 250mM, about 80mM to about 200mM, about 90mM to about 175mM, or about 100mM to about 150mM. In certain embodiments, TrisHCl wash buffer and / or elution buffer may also include a salt of about 75mM to about 100mM, about 100mM to about 125mM, about 125mM to about 150mM, about 150mM to about 175mM, about 175mM to about 200mM, about 200mM to about 225mM, or about 225mM to about 250mM salt. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 150 mM or 150 mM salt. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 125 mM or 125 mM salt.

[0121] In certain embodiments, the TrisHCl washing buffer and / or elution buffer may also include about 50mM to about 500mM NaCl. In certain embodiments, the TrisHCl washing buffer and / or elution buffer may also include about 55mM to about 400mM, about 60mM to about 350mM, about 70mM to about 300mM, about 75mM to about 250mM, about 80mM to about 200mM, about 90mM to about 175mM, or about 100mM to about 150mM NaCl. In certain embodiments, the TrisHCl washing buffer and / or elution buffer may also include about 75mM to about 100mM, about 100mM to about 125mM, about 125mM to about 150mM, about 150mM to about 175mM, about 175mM to about 200mM, about 200mM to about 225mM, or about 225mM to about 250mM NaCl. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 150 mM or 150 mM NaCl. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 125 mM or 125 mM NaCl.

[0122] In certain embodiments, the TrisHCl wash buffer and / or elution buffer may also include about 10% to about 75% (w / w) ethylene glycol. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may also include about 20% to about 72%, about 25% to about 70%, about 30% to about 65%, or about 40% to about 60% (w / w) ethylene glycol. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may also include about 30% to about 35%, about 35% to 40%, about 40% to about 45%, about 45% to about 50%, about 48% to about 52%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, or about 70% to about 75% (w / w) ethylene glycol. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may include about 50% or 50% (w / w) ethylene glycol.

[0123] In certain embodiments, TrisHCl wash buffer and / or elution buffer may also include an organic solvent or detergent. In some embodiments, the organic solvent or detergent is polysorbate 80 (e.g., Tween 80 or Crillet). In certain embodiments, polysorbate 80 may be about 0.0005% to about 5%, about 0.0005% to about 1%, about 0.001% to about 4%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.005% to about 3%, about 0.01% to about 2.5%, about 0.05% to about 5%, about 0.05% to about 2%, 0.05% to about 0.2%, or about 0.1% to about 1.5% (w / w) polysorbate 80. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 0.05% to about 0.08%, about 0.08% to about 0.11%, about 0.11% to about 0.14%, about 0.14% to about 0.17%, or about 0.17% to about 0.20% (w / w) polysorbate 80. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 0.1% or 0.1% (w / w) polysorbate 80. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 0.005% or 0.005% (w / w) polysorbate 80. In certain embodiments, the TrisHCl wash buffer and / or elution buffer may contain about 0.1% (w / w) polysorbate 80.

[0124] In certain embodiments, the pH of the TrisHCl wash buffer and / or elution buffer may be about 7.5 to about 9.2, about 8.0 to about 9.0, or about 8.0 to about 8.8. In certain embodiments, the pH of the TrisHCl wash buffer and / or elution buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the TrisHCl wash buffer and / or elution buffer may be about 7.4 or 7.4. In certain embodiments, the pH of the TrisHCl wash buffer and / or elution buffer may be about 8.5 or 8.5.

[0125] In certain embodiments, wash buffer and / or elution buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt. In certain embodiments, wash buffer and / or elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt. In certain embodiments, wash buffer and / or elution buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt. In certain embodiments, wash buffer and / or elution buffer may include about 50mM TrisHCl and about 125mM salt. In certain embodiments, wash buffer and / or elution buffer may include 50mM TrisHCl and 125mM salt. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, or about 8.0 to about 8.8. In certain embodiments, the pH of the buffer is about 8.5 or 8.5.

[0126] In certain embodiments, wash buffer and / or elution buffer may comprise about 10mM to about 200mM TrisHCl and about 10% to about 75% (w / w) ethylene glycol. In certain embodiments, wash buffer and / or elution buffer may comprise about 25mM to about 100mM TrisHCl and about 25% to about 70% (w / w) ethylene glycol. In certain embodiments, wash buffer and / or elution buffer may comprise about 40mM to about 60mM TrisHCl and about 40% to about 60% (w / w) ethylene glycol. In certain embodiments, wash buffer and / or elution buffer may comprise about 50mM TrisHCl and about 50% (w / w) ethylene glycol. In certain embodiments, wash buffer and / or elution buffer may comprise 50mM TrisHCl and 50% (w / w) ethylene glycol. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, or about 8.0 to about 8.8. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 8.5 or 8.5.

[0127] In certain embodiments, the wash buffer and / or elution buffer may comprise about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate. In certain embodiments, the wash buffer and / or elution buffer may comprise about 20 mM TrisHCl, about 150 mM salt, and about 0.1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise 20 mM TrisHCl, 150 mM salt, and 0.1% (w / w) polysorbate 80. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the pH of the wash buffer and / or elution buffer may be about 6.0 to about 8.8, about 6.5 to about 8.5, or about 7.0 to about 8.0. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 7.4 or 7.4.

[0128] In certain embodiments, the TrisHCl buffer is the first wash buffer and the elution buffer. In certain embodiments, the TrisHCl buffer is the first wash buffer, the third wash buffer and the elution buffer. In certain embodiments, the TrisHCl buffer is the second wash buffer and the elution buffer. In certain embodiments, the TrisHCl buffer is the second wash buffer, the fourth wash buffer and the elution buffer. In certain embodiments, the TrisHCl buffer is the second wash buffer, but not the elution buffer. In certain embodiments, the TrisHCl buffer is the second wash buffer and the fourth wash buffer, but not the elution buffer. About 5× column volumes or 5× column volumes of each wash buffer can be used. About 10× column volumes or 10× column volumes of each wash buffer can be used. About 5× column volumes or 5× column volumes of elution buffer can be used. About 10× column volumes or 10× column volumes of elution buffer can be used.

[0129] In certain embodiments, the wash buffer and / or elution buffer comprises about 10 mM to about 2000 mM sodium acetate. In certain embodiments, the wash buffer and / or elution buffer comprises about 20 mM to about 1000 mM, about 30 to about 750 mM, about 40 mM to about 500 mM, about 50 mM to about 200 mM, about 75 mM to about 175 mM, about 80 mM to about 150 mM, about 85 mM to about 125 mM, or about 90 mM to about 110 mM sodium acetate. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 90 mM to about 110 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 400 mM, or about 500 mM to about 600 mM. about 350 mM, about 350 to about 400 mM, about 400 to about 450 mM, about 450 mM to about 500 mM, about 500 to about 550 mM, about 550 to about 600 mM, about 600 to about 650 mM, about 650 to about 700 mM, about 700 to about 750 mM, about 750 to about 800 mM, about 800 mM to about 850 mM, about 850 mM to about 900 mM, about 900 to about 950 mM, about 1000 to about 10 ... 0mM, about 950mM to about 1000mM, about 1000 to about 1050mM, about 1050 to about 1100mM, about 1100 to about 1150mM, about 1150 to about 1200mM, about 1200 to about 1250mM, about 1250 to about 1300mM, about 1300 to about 1350mM, about 1350 to about 1400mM, about 1400 to about 1450mM, about 1450 to about In some embodiments, the wash buffer and / or elution buffer may comprise about 100 mM or 100 mM sodium acetate.

[0130] In certain embodiments, the wash buffer and / or elution buffer may comprise about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about 500mM, about 525mM, about 550mM, about 575mM, about 600mM, about 625mM, about 650mM, about 675mM, about 700mM, about 725mM, about 750mM, about 775mM, about 800mM, about 825mM, about 850mM, about 875mM, about 900mM, about 925mM, about 950mM, about 975mM, about 1000mM, about 1025mM, about 1 050mM, about 1075mM, about 1100mM, about 1125mM, about 1150mM, about 1175mM, about 1200mM, about 1225mM, about 1250mM, about 1275mM, about 1300mM, about 1325mM, about 1350mM, about 1375mM, about 1400mM, about 1425mM, about 1450mM, about 1475mM, about 1500mM, about 1525mM In some embodiments, the wash buffer and / or elution buffer may comprise about 100 mM or 100 mM sodium acetate.

[0131] In certain embodiments, wash buffer and / or elution buffer may include about 50mM to about 200mM sodium acetate. In certain embodiments, wash buffer and / or elution buffer may include about 50mM to about 80mM, about 70mM to about 100mM, about 80mM to about 110mM, about 90mM to about 120mM, about 100mM to about 130mM, about 120mM to about 150mM, about 140mM to about 170mM, or about 170mM to about 200mM sodium acetate. In certain embodiments, wash buffer and / or elution buffer may include about 60, about 70mM, about 80mM, about 90mM, about 100mM, about 110mM, about 120mM, about 130mM, about 140mM, about 150mM or about 160mM sodium acetate.

[0132] In certain embodiments, sodium acetate wash buffer and / or elution buffer may also include an organic solvent or detergent. In some embodiments, the organic solvent or detergent is polysorbate 80 (e.g., Tween 80 or Crillet). In certain embodiments, polysorbate 80 may be about 0.0005% to about 5%, about 0.0005% to about 1%, about 0.001% to about 4%, about 0.001% to about 0.1%, about 0.001% to about 0.05%, about 0.005% to about 3%, about 0.01% to about 2.5%, about 0.05% to about 5%, about 0.05% to about 2%, 0.05% to about 0.2%, or about 0.1% to about 1.5% (w / w) polysorbate 80. In certain embodiments, the sodium acetate buffer may comprise about 0.05% to about 0.08%, about 0.08% to about 0.11%, about 0.11% to about 0.14%, about 0.14% to about 0.17%, or about 0.17% to about 0.20% (w / w) polysorbate 80. In certain embodiments, the sodium acetate buffer may comprise about 0.1% or 0.1% (w / w) polysorbate 80. In certain embodiments, the sodium acetate buffer may comprise about 0.005% or 0.005% (w / w) polysorbate 80. In certain embodiments, the sodium acetate buffer may comprise about 0.1% (w / w) polysorbate 80.

[0133] In certain embodiments, the pH of the sodium acetate wash buffer and / or elution buffer may be about 5.0 to about 7.4, about 5.5 to about 7.0, or about 5.5 to about 6.5. In certain embodiments, the pH of the sodium acetate wash buffer and / or elution buffer may be about 5.2 to about 5.5, about 5.5 to about 5.8, about 5.8 to about 6.1, about 6.1 to about 6.4, or about 6.4 to about 6.8. In certain embodiments, the pH of the sodium acetate wash buffer and / or elution buffer is about 6.0 or 6.0.

[0134] In certain embodiments, the wash buffer and / or elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise 100 mM sodium acetate and 0.1% (w / w) polysorbate 80. In certain embodiments, the pH of the wash buffer and / or elution buffer may be about 5.0 to about 7.4, about 5.5 to about 7.0, or about 5.5 to about 6.5. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 6.0 or 6.0.

[0135] In certain embodiments, sodium acetate buffer is the first wash buffer and elution buffer. In certain embodiments, sodium acetate buffer is the first wash buffer, the third wash buffer and elution buffer. In certain embodiments, sodium acetate buffer is the second wash buffer and elution buffer. In certain embodiments, sodium acetate buffer is the second wash buffer, the fourth wash buffer and elution buffer. In certain embodiments, sodium acetate buffer is the second wash buffer, but not elution buffer. In certain embodiments, sodium acetate buffer is the second wash buffer and the fourth wash buffer, but not elution buffer.

[0136] About 5× column volumes or 5× column volumes of each sodium acetate wash buffer may be used. About 10× column volumes or 10× column volumes of each sodium acetate wash buffer may be used. About 5× column volumes or 5× column volumes of sodium acetate elution buffer may be used. About 10× column volumes or 10× column volumes of sodium acetate elution buffer may be used.

[0137] In certain embodiments, the wash buffer and / or elution buffer comprises about 10 mM to about 500 mM glycine. In certain embodiments, the wash buffer and / or elution buffer comprises about 10 mM to about 400 mM, about 10 mM to about 300 mM, about 10 mM to about 200 mM, about 15 mM to about 175 mM, about 20 mM to about 150 mM, about 25 mM to about 125 mM, about 25 mM to about 100 mM, about 30 mM to about 90 mM, about 35 mM to about 75 mM, or about 40 mM to about 60 mM glycine. In certain embodiments, the wash buffer and / or elution buffer comprises about 30 mM to about 35 mM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 50 mM to about 55 mM, about 55 mM to about 60 mM, about 60 mM to about 65 mM, about 65 mM to about 70 mM, about 70 mM to about 75 mM, about 75 mM to about 80 mM, about 80 mM to about 90 mM, about 100 mM to about 110 mM, about 120 mM to about 130 mM, about 140 mM to about 150 mM, about 160 mM to about 170 mM, about 180 mM to about 190 mM, about 190 mM to about 200 mM, about 180 mM to about 210 mM, about 190 mM to about 220 mM, about 190 mM to about 230 mM In some embodiments, the wash buffer and / or elution buffer may comprise about 50 mM or 50 mM glycine.

[0138] In certain embodiments, the wash buffer and / or elution buffer may comprise about 25 mM, about 50 mM, about 75 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 325 mM, about 350 mM, about 375 mM, about 400 mM, about 425 mM, about 450 mM, about 475 mM, about 500mM, about 525mM, about 550mM, about 575mM, about 600mM, about 625mM, about 650mM, about 675mM, about 700mM, about 725mM, about 750mM, about 775mM, about 800mM, about 825mM, about 850mM, about 875mM, about 900mM, about 925mM, about 950mM, about 975mM, about 1000mM, about 1025mM, about 1 050mM, about 1075mM, about 1100mM, about 1125mM, about 1150mM, about 1175mM, about 1200mM, about 1225mM, about 1250mM, about 1275mM, about 1300mM, about 1325mM, about 1350mM, about 1375mM, about 1400mM, about 1425mM, about 1450mM, about 1475mM, about 1500mM, about 1525mM In some embodiments, the wash buffer and / or elution buffer may comprise about 50 mM or 50 mM glycine.

[0139] In certain embodiments, the wash buffer and / or elution buffer comprises about 1 mM to about 300 mM histidine. In certain embodiments, the wash buffer and / or elution buffer comprises about 1 mM to about 250 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 1.5 mM to about 175 mM, about 2.0 mM to about 150 mM, about 2.5 mM to about 125 mM, about 2.5 mM to about 100 mM, about 3.0 mM to about 90 mM, about 3.5 mM to about 75 mM, about 4.0 to about 60 mM, about 5.0 mM to about 50 mM, about 6.0 mM to about 40 mM, about 7.0 to about 30 mM, about 8.0 mM to about 20 mM, or about 9.0 mM to about 15 mM histidine. In certain embodiments, the wash buffer and / or elution buffer comprises about 3.0 mM to about 3.5 mM, about 3.5 mM to about 4.0 mM, about 4.0 mM to about 4.5 mM, about 4.5 mM to about 5.0 mM, about 5.0 mM to about 5.5 mM, about 5.5 mM to about 6.0 mM, about 6.0 mM to about 6.5 mM, about 6.5 mM to about 7.0 mM, about 7.0 mM to about 7.5 mM, about 7.5 mM to about 8.0 mM, about 8.0 mM to about 9.0 mM, In some embodiments, the wash buffer and / or elution buffer may comprise about 10 mM or 10 mM histidine.

[0140] In certain embodiments, the wash buffer and / or elution buffer may comprise about 2.5 mM, about 5.0 mM, about 7.5 mM, about 10.0 mM, about 12.5 mM, about 15.0 mM, about 17.5 mM, about 20.0 mM, about 22.5 mM, about 25.0 mM, about 27.5 mM, about 30.0 mM, about 32.5 mM, about 35.0 mM, about 37.5 mM, about 40.0 mM, about 42.5 mM, about 45.0 mM, about 47.5 mM, about 5 0.0mM, about 52.5mM, about 55.0mM, about 57.5mM, about 60.0mM, about 62.5mM, about 65.0mM, about 67.5mM, about 70.0mM, about 72.5mM, about 75.0mM, about 77.5mM, about 80.0mM, about 82.5mM, about 85.0mM, about 87.5mM, about 90.0mM, about 92.5mM, about 95.0mM, about 97.5mM, about 100.0mM, about 102.5mM, about 105.0mM, about 107.5mM, about 110.0mM, about 112.5mM, about 115.0mM, about 117.5mM, about 120.0mM, about 122.5mM, about 125.0mM, about 127.5mM, about 130.0mM, about 132.5mM, about 135.0mM, about 137.5mM, about 140.0mM, about 142.5mM, about 145.0mM, about 147.5mM, about 150.0mM, about 152.5 In some embodiments, the wash buffer and / or elution buffer may comprise about 10 mM or 10 mM histidine.

[0141] In certain embodiments, the wash buffer and / or elution buffer may also contain about 1% to about 75% (w / w) trehalose. In certain embodiments, the wash buffer and / or elution buffer may also contain about 2% to about 50%, about 2.5% to about 25%, about 3.0% to about 20%, or about 4.0% to about 10% (w / w) trehalose. In certain embodiments, the wash buffer and / or elution buffer may also contain about 3.0% to about 3.5%, 3.5% to about 4.0%, about 4.0% to about 4.5%, about 4.5% to about 5.0%, about 4.8% to about 5.2%, about 5.0% to about 5.5%, about 5.5% to about 6.0%, about 6.0% to about 6.5%, about 6.5% to about 7.0%, or about 7.0% to about 7.5%, about 7.5% to about 8.0%, about 8.0% to about 8.5%, about 8.5% to about 9.0%, about 9.0% to about 9.5%, about 9.5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 25%, about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75% (w / w) trehalose. In certain embodiments, the wash buffer and / or elution buffer may contain about 5.0% or 5.0% (w / w) trehalose.

[0142] In certain embodiments, wash buffer and / or elution buffer may also include an organic solvent or detergent. In some embodiments, the organic solvent or detergent is polysorbate 80 (e.g., Tween 80 or Crillet). In certain embodiments, polysorbate 80 may be about 0.0005% to about 5%, about 0.0005% to about 1%, about 0.0006% to about 4%, about 0.0007% to about 0.1%, about 0.0008% to about 0.05%, about 0.0009% to about 3%, about 0.001% to about 2.5%, about 0.002% to about 5%, about 0.003% to about 2%, or 0.004% to about 0.2% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 0.05% to about 0.08%, about 0.08% to about 0.11%, about 0.11% to about 0.14%, about 0.14% to about 0.17%, or about 0.17% to about 0.20% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 0.1% or 0.1% (w / w) polysorbate 80. In some embodiments, polysorbate 80 may comprise about 0.005% or 0.005% (w / w) polysorbate 80. In some embodiments, polysorbate 80 is 0.005%.

[0143] In certain embodiments, the pH of the wash buffer and / or elution buffer may be from about 5.0 to about 9.0, from about 5.5 to about 8.0, or from about 6.0 to about 7.5. In certain embodiments, the pH of the wash buffer and / or elution buffer may be from about 5.2 to about 5.5, from about 5.5 to about 5.8, from about 5.8 to about 6.1, from about 6.1 to about 6.4, from about 6.4 to about 6.8, from about 6.8 to about 7.0, from about 7.0 to about 7.2, from about 7.2 to about 7.4, from about 7.4 to about 7.8, from about 7.8 to about 8.0, from about 8.0 to about 8.2, from about 8.2 to about 8.4, from about 8.4 to about 8.6, from about 8.6 to about 8.8, or from about 8.8 to about 9.0. In certain embodiments, the pH of the wash buffer and / or elution buffer is about 7.0 or 7.0.

[0144] In certain embodiments, the wash buffer and / or elution buffer may comprise about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% trehalose, and about 0.0005% to about 1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% trehalose, and about 0.001% to about 0.1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% trehalose, and about 0.001% to about 0.05% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% trehalose, and about 0.005% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise 50 mM glycine, 10 mM histidine, 100 mM salt, 5% trehalose, and 0.005% (w / w) polysorbate 80. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the pH of the buffer is about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.4. In certain embodiments, the pH of the buffer is about 7.0 to about 7.4.

[0145] In certain embodiments, the wash buffer and / or elution buffer may comprise about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM NaCl, about 1% to about 10% trehalose, and about 0.0005% to about 1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM NaCl, about 3% to about 8% trehalose, and about 0.001% to about 0.1% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM NaCl, about 4% to about 6% trehalose, and about 0.001% to about 0.05% (w / w) polysorbate 80. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM glycine, about 10 mM histidine, about 100 mM NaCl, about 5% trehalose, and about 0.005% (w / w) polysorbate 80. In certain embodiments, the elution buffer may comprise 50 mM glycine, 10 mM histidine, 100 mM NaCl, 5% trehalose, and 0.005% (w / w) polysorbate 80 at a pH of about 7.5, or about 7.0 to about 7.4. In certain embodiments, the pH of the buffer is about 7.0 to about 7.4.

[0146] In certain embodiments, the glycine buffer is the first wash buffer and elution buffer. In certain embodiments, the glycine buffer is the first wash buffer, the third wash buffer and elution buffer. In certain embodiments, the glycine buffer is the second wash buffer and elution buffer. In certain embodiments, the glycine buffer is the second wash buffer, the fourth wash buffer and elution buffer. In certain embodiments, the glycine buffer is the second wash buffer, but not the elution buffer. In certain embodiments, the glycine buffer is the second wash buffer and the fourth wash buffer, but not the elution buffer.

[0147] About 5× column volumes or 5× column volumes of each glycine wash buffer may be used. About 10× column volumes or 10× column volumes of each glycine wash buffer may be used. About 5× column volumes or 5× column volumes of glycine elution buffer may be used. About 10× column volumes or 10× column volumes of glycine elution buffer may be used.

[0148] In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM to about 500 mM sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 mM to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO, and tri(n-butyl)phosphate (TNBP). In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM to about 75 mM, about 75 mM to about 100 mM, about 90 mM to about 110 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 350 mM, about 350 to about 400 mM, about 400 to about 450 mM, or about 450 mM to about 500 mM MES-Na sodium salt. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM, about 75 mM, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM sodium salt of MES-Na.

[0149] In certain embodiments, wash buffer and / or elution buffer may comprise about 50mM to about 200mM taurine. In certain embodiments, wash buffer and / or elution buffer may comprise about 50mM to about 75mM, about 75mM to about 100mM, about 90mM to about 110mM, about 100mM to about 125mM, about 125mM to about 150mM, about 150mM to about 175mM, or about 175mM to about 200mM taurine. In certain embodiments, wash buffer and / or elution buffer may comprise about 50mM, about 75mM, about 90mM, about 100mM, about 125mM, about 150mM, about 175mM or about 200mM taurine.

[0150] In certain embodiments, the wash buffer and / or elution buffer may comprise about 80 mM to about 400 mM Bis-Tris. In certain embodiments, the wash buffer and / or elution buffer may comprise about 80 mM to about 100 mM, about 90 mM to about 110 mM, about 100 mM to about 125 mM, about 125 mM to about 150 mM, about 150 mM to about 175 mM, about 175 mM to about 200 mM, about 200 to about 250 mM, about 250 mM to about 300 mM, about 300 mM to about 350 mM, or about 350 to about 400 mM Bis-Tris. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50 mM, about 75 mM, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, or about 400 mM Bis-Tris.

[0151] The wash buffer and / or elution buffer may comprise about 30mM to about 35mM, about 35mM to about 40mM, about 40mM to about 45mM, about 45mM to about 50mM, about 50mM to about 55mM, about 55mM to about 60mM, about 60mM to about 65mM, about 65mM to about 70mM, about 70mM to about 75mM, or about 75mM to about 80mM arginine-HCl. In certain embodiments, the wash buffer and / or elution buffer may comprise about 50mM or 50mM arginine-HCl. In certain embodiments, the wash buffer and / or elution buffer may comprise about 75mM to about 100mM, about 100mM to about 125mM, about 125mM to about 150mM, about 150mM to about 175mM, about 175mM to about 200mM, about 200mM to about 225mM, or about 225mM to about 250mM NaCl. In certain embodiments, the wash buffer and / or elution buffer may comprise about 150mM or 150mM NaCl. In certain embodiments, the pH of the second buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In certain embodiments, the pH of the wash buffer and / or elution buffer may be about 8.5 or 8.5.

[0152] In some embodiments, the washing buffer and / or elution buffer may include about 50mM to about 200mM glycine. In some embodiments, the washing buffer and / or elution buffer may include about 50mM to about 100mM, about 70mM to about 120mM, about 100mM to about 150mM, about 120mM to about 170mM, about 150mM to about 200mM glycine. In some embodiments, the pH of the washing buffer and / or elution buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In some embodiments, the pH of the washing buffer and / or elution buffer may be about 8.5 or 8.5.

[0153] In some embodiments, the washing buffer and / or elution buffer may include about 50mM to about 20mM sodium citrate. In some embodiments, the second buffer may include about 5mM to about 10mM, about 7mM to about 12mM, about 10mM to about 15mM, about 12mM to about 17mM, or about 15mM to about 20mM sodium citrate. In some embodiments, the pH of the washing buffer and / or elution buffer may be about 7.5 to about 7.7, about 7.7 to about 7.9, about 7.9 to about 8.1, about 8.1 to about 8.3, about 8.3 to about 8.5, about 8.5 to about 8.7, about 8.7 to about 8.9, or about 8.9 to about 9.2. In some embodiments, the pH of the washing buffer and / or elution buffer may be about 8.5 or 8.5.

[0154] Washing buffer and / or elution buffer may comprise about 20mM to about 100mM histidine and about 75mM to about 250mM NaCl, with a pH of about 7.5 to about 8.8. In certain embodiments, washing buffer and / or elution buffer may comprise about 20mM to about 40mM, about 40mM to about 60mM, about 60mM to about 75mM, or about 75mM to about 100mM histidine. In certain embodiments, washing buffer and / or elution buffer may comprise about 20mM or 20mM histidine. In certain embodiments, wash buffer and / or elution buffer may comprise about 75mM to about 100mM, about 100mM to about 125mM, about 125mM to about 150mM, about 150mM to about 175mM, about 175mM to about 200mM, about 200mM to about 225mM NaCl, or about 225mM to about 250mM NaCl. In certain embodiments, wash buffer and / or elution buffer may comprise about 150mM or 150mM NaCl. In certain embodiments, the pH of wash buffer and / or elution buffer may be about 7.5 to about 7.9, about 7.8 to about 8.2, about 8.1 to about 8.5, about 8.4 to about 8.9, or about 8.6 to about 9.0. In certain embodiments, the pH of wash buffer and / or elution buffer may be about 8.0 or 8.0.

[0155] The wash buffer and / or elution buffer may comprise about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol at a pH of about 7.5 to about 9.2. The wash buffer and / or elution buffer may comprise about 20 mM to about 80 mM arginine-HCl and about 50 mM to about 200 mM salt at a pH of about 7.3 to about 8.8. The wash buffer and / or elution buffer may comprise about 50 mM TrisHCl and about 50% (w / w) ethylene glycol at a pH of about 8.5. The wash buffer and / or elution buffer may comprise about 20 mM to about 150 mM taurine, about 30% to about 75% (w / w) ethylene glycol, and 0.05% to 0.2% (w / w) octylpyranoglucoside at a pH of about 7.3 to about 8.8. The washing buffer and / or elution buffer may comprise about 50 mM to about 200 mM arginine-HCl, about 50 mM to about 200 mM lysine-HCl, about 50 mM to about 200 mM histidine-HCl, about 1 mM to about 4 mM N-acetyl-D, L-tryptophan, and about 10% to about 40% (w / w) polysorbate 80, with a pH of about 7.3 to about 8.8. In certain embodiments, if a salt (e.g., NaCl) is present in the washing buffer and / or elution buffer, the concentration of the salt does not exceed 500 mM in certain embodiments, and the concentration of the salt does not exceed 200 mM in certain embodiments. In certain embodiments, the salt is NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, or a combination of more than one of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate. In certain embodiments, the salt is NaCl. In certain embodiments, one or more of sorbitol, mannitol, xylitol, sucrose, or trehalose may be used in combination with or in place of ethylene glycol.

[0156] In certain embodiments, the first washing step uses a first buffer, which may be a buffer based on TrisHCl. In certain embodiments, the first washing step uses a first buffer, which may be a buffer based on sodium acetate (NaAc). In certain embodiments, the first washing step uses a first buffer, which includes a sodium salt (MES-Na) of 2-(N-morpholino) ethanesulfonic acid, EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, the first washing step uses a first buffer, which includes about 50mM to about 200mM taurine and 0.2% to 1.5% PEG (e.g., PEG6000). In certain embodiments, the first washing step uses a first buffer, which includes Bis-Tris and a solvent / detergent mixture containing Triton-X 100, polysorbate 80, and TNBP. In certain embodiments, the first washing step uses a first buffer, which includes sodium acetate and polysorbate 80. One or more or even all of these washing steps are performed at room temperature. In certain embodiments, the first washing step uses a first buffer, which may be a TrisHCl-based buffer comprising NaCl.

[0157] In certain embodiments, the second washing step uses a second buffer, and the second buffer can be a buffer based on TrisHCl. In certain embodiments, the second washing step uses a second buffer, and the second buffer can be a buffer based on sodium acetate (NaAc). In certain embodiments, the second washing step uses a second buffer, and the second buffer includes a sodium salt (MES-Na) of 2-(N-morpholino) ethanesulfonic acid, EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, the second washing step uses a second buffer, and the second buffer includes about 50mM to about 200mM taurine and 0.2% to 1.5% PEG (such as PEG6000). In certain embodiments, the second washing step uses a second buffer, and the second buffer includes Bis-Tris and a solvent / detergent mixture containing Triton-X 100, polysorbate 80, and TNBP. In certain embodiments, the second washing step uses a second buffer, and the second buffer includes sodium acetate and polysorbate 80. In certain embodiments, the second washing step uses a second buffer comprising TrisHCl and NaCl buffer.

[0158] In certain embodiments, at least one wash buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, at least one wash buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, at least one wash buffer may comprise about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, at least one wash buffer may comprise about 50mM TrisHCl and about 125mM salt, with a pH of about 8.5. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. One or more of these washing steps or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0159] In certain embodiments, at least one wash buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, at a pH of about 5.0 to about 7.4. In certain embodiments, at least one wash buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, at a pH of about 5.5 to about 7.0. In certain embodiments, at least one wash buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, at a pH of about 5.5 to about 6.5. In certain embodiments, at least one wash buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, at a pH of about 6.0. One or more of these wash steps or even all of these wash steps are performed at room temperature. In certain embodiments, the wash steps are performed at room temperature.

[0160] In certain embodiments, at least two washing buffers are used. In certain embodiments, at least one washing buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, and pH is about 7.5 to about 9.2. In certain embodiments, at least one washing buffer may include about 10mM to about 2000mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, and pH is about 5.0 to about 7.4. In certain embodiments, salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate or a combination thereof. More than one of these washing steps or even all of these washing steps are carried out at room temperature. In certain embodiments, salt is NaCl. In certain embodiments, washing steps are carried out at room temperature.

[0161] In certain embodiments, at least two washing buffers are used. In certain embodiments, at least one washing buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and pH is about 8.0 to about 9.0. In certain embodiments, at least one washing buffer may include about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, and pH is about 5.5 to about 7.0. In certain embodiments, salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate or a combination thereof. More than one of these washing steps or even all of these washing steps are carried out at room temperature. In certain embodiments, salt is NaCl. In certain embodiments, washing steps are carried out at room temperature.

[0162] In certain embodiments, at least two washing buffers are used. In certain embodiments, at least one washing buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, and pH is about 8.0 to about 9.0. In certain embodiments, at least one washing buffer may include about 90mM to about 100mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and pH is about 5.5 to about 6.5. In certain embodiments, salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate or a combination thereof. More than one of these washing steps or even all of these washing steps are carried out at room temperature. In certain embodiments, salt is NaCl. In certain embodiments, washing steps are carried out at room temperature.

[0163] In certain embodiments, at least two washing buffers are used. In certain embodiments, at least one washing buffer may include about 50mM TrisHCl and about 125mM salt, and pH is about 8.5. In certain embodiments, at least one washing buffer may include about 100mM sodium acetate and about 0.1% (w / w) polysorbate 80, and pH is about 6.0. In certain embodiments, salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate or a combination thereof. More than one or even all of these washing steps are performed at room temperature. In certain embodiments, salt is NaCl. In certain embodiments, washing steps are performed at room temperature.

[0164] In some embodiments, at least 3 washing steps are performed; wherein at least one washing buffer comprises about 50 mM to about 2000 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.2 to about 6.8; at least one washing buffer comprises about 30 mM to about 200 mM TrisHCl and about 75 mM to about 500 mM salt, with a pH of about 7.5 to about 9.2; and at least one washing buffer comprises about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, with a pH of about 7.3 to about 8.8. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. One or more of these washing steps or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0165] In some embodiments, at least 3 washing steps are performed. In some embodiments, at least one buffer comprises about 100 mM sodium acetate, about 0.1% (w / w) polysorbate 80, and a pH of about 6.0. In some embodiments, at least one buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and a pH of about 8.5. In some embodiments, at least one buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and a pH of about 8.5. More than one or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0166] In some embodiments, at least three washing steps are performed; wherein the first washing step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 2000 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and the pH of the first buffer is about 5.2 to about 6.8; the second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 30 mM to about 200 mM TrisHCl and about 75 mM to about 500 mM salt, and the pH of the second buffer is about 7.5 to about 9.2; the third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 7.3 to about 8.8. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. One or more of these washing steps or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0167] In some embodiments, the method further comprises performing a fourth washing step before the first washing step, the fourth washing step comprising adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10 mM to about 30 mM TrisHCl and about 75 mM to about 250 mM NaCl, and the pH of the fourth buffer is about 6.5 to about 8.0.

[0168] In some embodiments, at least 3 washing steps are performed; wherein the first buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH of the third buffer is about 8.5. One or more of these washing steps or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0169] In some embodiments, at least three washing steps are performed; the first washing step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and the pH of the first buffer is about 5.5 to about 6.5; the second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 10 mM to about 70 mM TrisHCl and about 75 mM to about 250 mM NaCl, and the pH of the second buffer is about 8.0 to about 9.0; the third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 10 mM to about 70 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 8.0 to about 9.0. In some embodiments, the method further comprises performing a fourth washing step prior to the first washing step, the fourth washing step comprising adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10 mM to about 30 mM TrisHCl and about 75 mM to about 250 mM NaCl, the pH of the fourth buffer being about 6.5 to about 8.0. More than one or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0170] In some embodiments, at least three washing steps are performed; the first washing step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and the pH of the first buffer is about 5.5 to about 6.5; the second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 10 mM to about 70 mM TrisHCl and about 75 mM to about 250 mM NaCl, and the pH of the second buffer is about 8.0 to about 9.0; the third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 10 mM to about 70 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 8.0 to about 9.0. In some embodiments, the method further comprises performing a fourth washing step prior to the first washing step, the fourth washing step comprising adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10 mM to about 30 mM TrisHCl and about 75 mM to about 250 mM NaCl, the pH of the fourth buffer being about 6.5 to about 8.0. More than one or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0171] In some embodiments, the first buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH of the first buffer is about 6.0. In some embodiments, the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and the pH of the second buffer is about 8.5. In some embodiments, the third buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH of the third buffer is about 8.0. More than one or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0172] In even more embodiments, at least three washing steps are performed; the first washing step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 500 mM 2-(N-morpholino)ethanesulfonic acid sodium salt (MES-Na), about 3 mM to about 30 mM EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP), the first buffer having a pH of about 5.2 to about 6.8; the second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 30 mM to about 200 mM TrisHCl or arginine-HCl and about 75 mM to about 500 mM salt, the second buffer having a pH of about 7.5 to about 9.2; the third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 20 mM to about 80 mM arginine-HCl and about 50% to about 60% (w / w) sucrose, the third buffer having a pH of about 7.3 to about 8.8. In some embodiments, the salt is selected from: NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of more than one of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl and sodium acetate. In some embodiments, the salt is NaCl. In some embodiments, the concentration of the salt does not exceed 500mM. In some embodiments, the concentration of the salt does not exceed 200mM. In some embodiments, the concentration of the salt in the third buffer does not exceed 500mM. In some embodiments, the concentration of the salt in the third buffer does not exceed 200mM. In some embodiments, the method also includes performing a fourth washing step before the first washing step, the fourth washing step including adding a fourth buffer to the affinity resin, the fourth buffer comprising about 20mM to about 100mM arginine-HCl and about 75mM to about 250mM NaCl, and the pH of the fourth buffer is about 7.5 to about 8.8. More than one of these washing steps or even all of these washing steps are performed at room temperature. In certain embodiments, the washing steps are performed at room temperature.

[0173] In some embodiments, the method further comprises a washing step after the first elution step and before the second elution step, the washing step comprising adding a fifth buffer to the affinity resin, the fifth buffer comprising about 20mM to about 100mM arginine-HCl and about 75mM to about 250mM NaCl, the pH of the fifth buffer being about 7.5 to about 8.5. In some embodiments, the second elution step comprises adding a second elution buffer to the affinity resin, the second elution buffer comprising about 20mM to about 100mM arginine-HCl, about 40% to about 60% (w / w) glycerol and about 500mM to 1000mM salt (e.g., NaCl), the pH of the second elution buffer being about 7.5 to about 8.5.

[0174] The additional washing step can effectively minimize the fronting effect between the first elution step and the second elution step, for example, providing an elution triggered only by the first elution buffer and the second elution buffer themselves, rather than an elution triggered by a front that may be caused by a mixture of the first elution buffer and the second elution buffer. In some embodiments, the method further includes performing a sixth washing step after the fifth washing step and the second elution step, the sixth washing step comprising adding a sixth buffer to the affinity resin, the sixth buffer comprising about 20 mM to about 100 mM arginine-HCl and about 75 mM to about 250 mM NaCl, and the pH of the sixth buffer is about 7.5 to about 8.5.

[0175] In some embodiments, at least three washing steps are performed; the first washing step comprises adding a first buffer to the affinity resin, the first buffer comprising about 80 mM to about 400 mM Bis-Tris and about 10 grams to about 20 grams of a solvent / detergent mixture, the solvent / detergent mixture comprising Triton-X 100, polysorbate 80, and TNBP in a ratio of about 11:3:3 (by weight), the first buffer having a pH of about 5.2 to about 6.8; the second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 5 mM to about 20 mM sodium citrate, the second buffer having a pH of about 7.5 to about 9.2; the third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 50 mM to about 200 mM arginine-HCl, about 50 mM to about 200 mM lysine-HCl, about 50 mM to about 200 mM histidine-HCl, about 1 mM to about 4 mM N-acetyl-D,L-tryptophan and about 10% to about 40% (w / w) polysorbate 80, the third buffer having a pH of about 7.3 to about 8.8.

[0176] In certain embodiments, the first elution step uses a first buffer, which may be a buffer based on TrisHCl. In certain embodiments, the first elution step uses a first buffer, which may be a buffer based on sodium acetate (NaAc). In certain embodiments, the first elution step uses a first buffer, which comprises a sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, the first elution step uses a first buffer, which comprises about 50mM to about 200mM taurine and 0.2% to 1.5% PEG (e.g., PEG6000). In certain embodiments, the first elution step uses a first buffer, which comprises Bis-Tris and a solvent / detergent mixture containing Triton-X 100, polysorbate 80, and TNBP. In certain embodiments, the first elution step uses a first buffer, which comprises sodium acetate and polysorbate 80. One or more or even all of these elution steps are performed at room temperature. In certain embodiments, the first elution step uses a first buffer, which may be a TrisHCl-based buffer comprising NaCl.

[0177] In certain embodiments, the second elution step uses a second buffer, and the second buffer can be a buffer based on TrisHCl. In certain embodiments, the second elution step uses a second buffer, and the second buffer can be a buffer based on sodium acetate (NaAc). In certain embodiments, the second elution step uses a second buffer, and the second buffer includes a sodium salt (MES-Na) of 2-(N-morpholino) ethanesulfonic acid, EDTA, and a solvent / detergent mixture containing polysorbate 80, DMSO, and tri(n-butyl) phosphate (TNBP). In certain embodiments, the second elution step uses a second buffer, and the second buffer includes about 50mM to about 200mM taurine and 0.2% to 1.5% PEG (such as PEG6000). In certain embodiments, the second elution step uses a second buffer, and the second buffer includes Bis-Tris and a solvent / detergent mixture containing Triton-X 100, polysorbate 80, and TNBP. In certain embodiments, the second elution step uses a second buffer, and the second buffer includes sodium acetate and polysorbate 80. In certain embodiments, the second elution step uses a second buffer comprising TrisHCl and NaCl buffer.

[0178] In certain embodiments, at least one elution buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, at least one elution buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, at least one elution buffer may comprise about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, at least one elution buffer may comprise about 50mM TrisHCl and about 125mM salt, with a pH of about 8.5. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0179] In certain embodiments, at least one elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, at least one elution buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, at least one elution buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, at least one elution buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0180] In certain embodiments, at least one elution buffer may comprise about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% (w / w) trehalose, and about 0.0005% to about 1% (w / w) polysorbate 80, at a pH of about 6.0 to about 8.0. In certain embodiments, at least one elution buffer may comprise about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% (w / w) trehalose, and about 0.001% to about 0.1% (w / w) polysorbate 80, at a pH of about 6.5 to about 7.5. In certain embodiments, at least one elution buffer may comprise about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% (w / w) trehalose, and about 0.001% to about 0.05% (w / w) polysorbate 80, with a pH of about 7.0 to about 7.4. In certain embodiments, at least one elution buffer may comprise about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose, and about 0.005% (w / w) polysorbate 80, with a pH of about 7.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0181] In certain embodiments, at least one elution buffer may comprise about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 6.0 to about 8.8. In certain embodiments, at least one elution buffer may comprise about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 6.5 to about 8.5. In certain embodiments, at least one elution buffer may comprise about 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 7.0 to about 8.0. In certain embodiments, at least one elution buffer may comprise about 20 mM TrisHCl, about 150 mM salt and about 0.1% (w / w) polysorbate 80, with a pH of about 7.4. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C or 2°C to 8°C, as described above).

[0182] In certain embodiments, at least one elution buffer may comprise about 10 mM to about 200 mM TrisHCl and about 10% to about 75% (w / w) ethylene glycol, with a pH of about 7.5 to about 9.2. In certain embodiments, at least one elution buffer may comprise about 25 mM to about 100 mM TrisHCl and about 25% to about 70% (w / w) ethylene glycol, with a pH of about 8.0 to about 9.0. In certain embodiments, at least one elution buffer may comprise about 40 mM to about 60 mM TrisHCl and about 40% to about 60% (w / w) ethylene glycol, with a pH of about 8.0 to about 8.8. In certain embodiments, at least one elution buffer may comprise about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, with a pH of about 8.5.

[0183] In certain embodiments, at least two elution buffers are used. In certain embodiments, at least one elution buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, and a pH of about 7.5 to about 9.2. In certain embodiments, at least one elution buffer may include about 10mM to about 2000mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, and a pH of about 5.0 to about 7.4. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0184] In certain embodiments, at least two elution buffers are used. In certain embodiments, at least one elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, at least one elution buffer may include about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, and a pH of about 5.5 to about 7.0. In certain embodiments, salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0185] In certain embodiments, at least two elution buffers are used. In certain embodiments, at least one elution buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, at least one elution buffer may include about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and a pH of about 5.5 to about 6.5. In certain embodiments, salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0186] In certain embodiments, at least two elution buffers are used. In certain embodiments, at least one elution buffer may include about 50mM TrisHCl and about 125mM salt, and the pH is about 8.5. In certain embodiments, at least one elution buffer may include about 100mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH is about 6.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0187] In some embodiments, at least three elution steps are performed; wherein at least one elution buffer comprises about 50 mM to about 2000 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.2 to about 6.8; at least one elution buffer comprises about 30 mM to about 200 mM TrisHCl and about 75 mM to about 500 mM salt, with a pH of about 7.5 to about 9.2; at least one elution buffer comprises about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, with a pH of about 7.3 to about 8.8. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0188] In some embodiments, at least 3 elution steps are performed. In some embodiments, at least one buffer comprises about 100 mM sodium acetate, about 0.1% (w / w) polysorbate 80, and a pH of about 6.0. In some embodiments, at least one buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and a pH of about 8.5. In some embodiments, at least one buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and a pH of about 8.5. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0189] In some embodiments, at least three elution steps are performed; wherein the first elution step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 2000 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and the pH of the first buffer is about 5.2 to about 6.8; the second elution step comprises adding a second buffer to the affinity resin, the second buffer comprising about 30 mM to about 200 mM TrisHCl and about 75 mM to about 500 mM salt, and the pH of the second buffer is about 7.5 to about 9.2; the third elution step comprises adding a third buffer to the affinity resin, the third buffer comprising about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 7.3 to about 8.8. In certain embodiments, the salt can be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0190] In some embodiments, at least one elution buffer has a pH of about 5.8 to about 6.2 and comprises about 90 mM to about 110 mM sodium acetate and about 0.09% to about 0.11% (w / w) polysorbate 80 / Tween 80. In some embodiments, at least one elution buffer has a pH of about 6.5 to about 7.5 and comprises about 35 mM to 70 mM glycine, 5 mM to 15 mM histidine, 50 mM to 200 mM NaCl, 3% to 8% trehalose, and 0.001% to 0.005% Crillet TM 4. In some embodiments, at least one elution buffer has a pH of about 8.2 to about 8.8 and comprises about 45 mM to about 55 mM TrisHCl and about 90 mM to about 150 mM NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0191] In some embodiments, the first elution buffer has a pH of about 5.8 to about 6.2 and comprises about 90 mM to about 110 mM sodium acetate and about 0.09% to about 0.11% (w / w) polysorbate 80 / Tween 80. In some embodiments, the second elution buffer has a pH of about 6.5 to about 7.5 and comprises about 35 mM to 70 mM glycine, 5 mM to 15 mM histidine, 50 mM to 200 mM NaCl, 3% to 8% trehalose, and 0.001% to 0.005% Crillet TM 4. In some embodiments, the third elution buffer has a pH of about 8.2 to about 8.8 and comprises about 45 mM to about 55 mM TrisHCl and about 90 mM to about 150 mM NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0192] In some embodiments, the pH of the first elution buffer is about 5.8 to about 6.2, and comprises about 90mM to about 110mM sodium acetate and about 0.09% to about 0.11% (w / w) polysorbate 80 / Tween 80; the pH of the second buffer is about 8.2 to about 8.8, and comprises about 45mM to about 55mM TrisHCl and about 110mM to about 135mM NaCl; the pH of the third buffer is about 8.2 to about 8.8, and comprises about 45mM to about 55mM TrisHCl and about 45% to about 55% ethylene glycol. In certain embodiments, there is an optional fourth buffer, the pH of the fourth buffer is about 7.2 to about 7.6, and comprises about 15mM to about 25mM TrisHCl and about 135mM to about 165mM NaCl. In certain embodiments, the elution buffer has a pH of about 7.8 to about 8.2 and comprises about 45 mM to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 mM to about 850 mM NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0193] In some embodiments, the pH of at least one elution buffer is about 7.2 to about 7.6, and comprises about 15mM to about 25mM TrisHCl and about 135mM to about 165mM NaCl. In some embodiments, the pH of at least one elution buffer is about 5.8 to about 6.2, and comprises about 90mM to about 110mM sodium acetate and about 0.09% to about 0.11% (w / w) polysorbate 80. In some embodiments, the pH of at least one elution buffer is about 8.2 to about 8.8, and comprises about 45mM to about 55mMTrisHCl and about 110mM to about 135mM NaCl. In some embodiments, the pH of at least one elution buffer is about 7.5 to about 8.5, and comprises about 45mM to about 55mM TrisHCl and about 45% to about 55% ethylene glycol. In some embodiments, at least one elution buffer has a pH of about 7.8 to about 8.2 and comprises about 45 mM to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 mM to about 850 mM NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0194] In some embodiments, the first buffer has a pH of about 7.2 to about 7.6 and comprises about 15 mM to about 25 mM TrisHCl and about 135 mM to about 165 mM NaCl; the second buffer has a pH of about 5.8 to about 6.2 and comprises about 90 mM to about 110 mM sodium acetate and about 0.09% to about 0.11% (w / w) polysorbate 80; the third buffer has a pH of about 8.2 to about 8.8 and comprises about 45 mM to about 55 mM TrisHCl and about 110 mM to about 135 mM NaCl; the fourth buffer has a pH of about 7.5 to about 8.5 and comprises about 45 mM to about 55 mM TrisHCl and about 45% to about 55% ethylene glycol; the fifth elution buffer has a pH of about 7.8 to about 8.2 and comprises about 45 mM to about 55 mM TrisHCl, about 45% to about 55% ethylene glycol, and about 650 mM to about 850 mM NaCl. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0195] In certain embodiments, the first washing buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the first elution buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0196] In certain embodiments, the first washing buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0197] In certain embodiments, the first washing buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 8.8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0198] In certain embodiments, the first washing buffer may comprise about 50mM TrisHCl and about 125mM salt, and the pH is about 8.5. In certain embodiments, the first elution buffer may comprise about 50mM TrisHCl and about 125mM salt, and the pH is about 8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0199] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the first elution buffer may comprise about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0200] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0201] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 8.8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0202] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 50mM TrisHCl and about 125mM salt, and the pH is about 8.5. In certain embodiments, the first elution buffer may comprise about 50mM TrisHCl and about 125mM salt, and the pH is about 8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0203] In certain embodiments, the first wash buffer and the third wash buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, and a pH of about 7.5 to about 9.2. In certain embodiments, the second wash buffer may include about 10mM to about 2000mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, and a pH of about 5.0 to about 7.4. In certain embodiments, the first elution buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, and a pH of about 7.5 to about 9.2. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0204] In certain embodiments, the first wash buffer and the third wash buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the second wash buffer may include about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, and a pH of about 5.5 to about 7.0. In certain embodiments, the first elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0205] In certain embodiments, the first wash buffer and the third wash buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the second wash buffer may include about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and a pH of about 5.5 to about 6.5. In certain embodiments, the first elution buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 8.8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0206] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 50mM TrisHCl and about 125mM salt, with a pH of about 8.5. In certain embodiments, the second wash buffer may comprise about 100mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the first elution buffer may comprise about 50mMTrisHCl and about 125mM salt, with a pH of about 8. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0207] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the second wash buffer and the fourth wash buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the first elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0208] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the second wash buffer and the fourth wash buffer may comprise about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the first elution buffer may comprise about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0209] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the second wash buffer and the fourth wash buffer may comprise about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the first elution buffer may comprise about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0210] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 50mM TrisHCl and about 125mM salt, and the pH is about 8.5. In certain embodiments, the second wash buffer and the fourth wash buffer may comprise about 100mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH is about 6.0. In certain embodiments, the first elution buffer may comprise about 100mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH is about 6.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0211] In certain embodiments, the first wash buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the first elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the elution step occurs at less than 18° C. (e.g., 1° C. to 12° C., or 2° C. to 8° C., as described above).

[0212] In certain embodiments, the first wash buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the first elution buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0213] In certain embodiments, the first wash buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the first elution buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0214] In certain embodiments, the first wash buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0. In certain embodiments, the first elution buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18° C. (e.g., 1° C. to 12° C., or 2° C. to 8° C., as described above).

[0215] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the first elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0216] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the first elution buffer may comprise about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0217] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the first elution buffer may comprise about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0218] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the first elution buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18° C. (e.g., 1° C. to 12° C., or 2° C. to 8° C., as described above).

[0219] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the second wash buffer may comprise about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt, with a pH of about 7.5 to about 9.2. In certain embodiments, the first elution buffer may comprise about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, with a pH of about 5.0 to about 7.4. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0220] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the second wash buffer may comprise about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may comprise about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, with a pH of about 5.5 to about 7.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0221] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the second wash buffer may comprise about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, with a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may comprise about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, with a pH of about 5.5 to about 6.5. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0222] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the second wash buffer may comprise about 50 mM TrisHCl and about 125 mM salt, with a pH of about 8.5. In certain embodiments, the first elution buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0223] In certain embodiments, the first wash buffer and the third wash buffer may include about 10mM to about 2000mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, and a pH of about 5.0 to about 7.4. In certain embodiments, the second wash buffer and the fourth wash buffer may include about 10mM to about 200mM TrisHCl and about 50mM to about 500mM salt, and a pH of about 7.5 to about 9.2. In certain embodiments, the first elution buffer may include about 10mM to about 200mMTrisHCl and about 50mM to about 500mM salt, and a pH of about 7.5 to about 9.2. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0224] In certain embodiments, the first wash buffer and the third wash buffer may include about 50mM to about 200mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80, and a pH of about 5.5 to about 7.0. In certain embodiments, the second wash buffer and the fourth wash buffer may include about 25mM to about 100mM TrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may include about 25mM to about 100mMTrisHCl and about 75mM to about 250mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0225] In certain embodiments, the first wash buffer and the third wash buffer may include about 90mM to about 110mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, and a pH of about 5.5 to about 6.5. In certain embodiments, the second wash buffer and the fourth wash buffer may include about 40mM to about 60mM TrisHCl and about 100mM to about 150mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the first elution buffer may include about 40mM to about 60mMTrisHCl and about 100mM to about 150mM salt, and a pH of about 8.0 to about 9.0. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(eg, 1°C to 12°C, or 2°C to 8°C, as described above).

[0226] In certain embodiments, the first wash buffer and the third wash buffer may comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, with a pH of about 6.0. In certain embodiments, the second wash buffer and the fourth wash buffer may comprise about 50 mM TrisHCl and about 125 mM salt, with a pH of about 8.5. In certain embodiments, the first elution buffer may comprise about 50 mM TrisHCl and about 125 mM salt, with a pH of about 8.5. In certain embodiments, the salt may be NaCl, KCl, MgCl2, CaCl2, LiCl, CsCl, sodium acetate (C2H3NaO2), (NH4)2SO4, NH4Cl, Na2SO4, K2SO4, sodium citrate, potassium citrate, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the washing step is performed at room temperature. In certain embodiments, the elution step occurs at less than 18°C ​​(e.g., 1°C to 12°C, or 2°C to 8°C, as described above).

[0227] Further examples of wash buffers and / or elution buffers can be found in WO2019133677, which is incorporated herein in its entirety for all intended purposes.

[0228] Resin

[0229] In some embodiments, the affinity resin is POROS TM CaptureSelect TM AAVx.

[0230] In some embodiments, the AAV is AAV8 and the affinity resin is POROS TM CaptureSelect TM In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM AAV9. In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM AAVx.

[0231] In some embodiments, the AAV is AAV8 and the affinity resin is POROS TM CaptureSelect TM AAV8, the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM AAV9, the elution buffer is acidic and does not contain ethylene glycol. In some embodiments, the AAV is AAV9 and the affinity resin is POROS TM CaptureSelect TM For AAVx, the elution buffer is acidic and does not contain ethylene glycol.

[0232] In some embodiments, the AAV is AAV8 and the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV8 from the ADK8 or ADK8 / 9 type immobilized on a chromatographic matrix. In some embodiments, the AAV is AAV9 and the affinity resin is an immunoaffinity resin consisting of an immobilized monoclonal antibody against AAV9 from the ADK9 or ADK8 / 9 type immobilized on a chromatographic matrix.

[0233] Source of rAAV particles

[0234] With regard to the methods disclosed herein, AAV can be any AAV serotype. In certain embodiments, the AAV purified by the methods described herein is AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, AAV11 serotype, AAV12 serotype, AAV13 serotype, AAAV serotype, BAAV serotype, AAV (VR-195) serotype and AAV (VR-355) serotype, or a chimeric AAV vector. In certain embodiments, AAV is wild type.

[0235] In some embodiments, the AAV is of the AAV5 serotype. In some embodiments, the AAV is of the AAV9 serotype. In some embodiments, the AAV is genetically engineered and / or chemically modified. In some embodiments, the AAV comprises a modified capsid, such as a genetically engineered or chemically modified AAV capsid. In some embodiments, the AAV vector comprises VP1, which comprises the sequence of SEQ ID NO: 1. In some embodiments, the AAV vector comprises VP2, which comprises the sequence of SEQ ID NO: 2. In some embodiments, the AAV vector comprises VP3, which comprises the sequence of SEQ ID NO: 3.

[0236] In some embodiments, AAV is genetically modified and / or chemically modified. In certain embodiments, AAV is genetically modified and / or chemically modified. In certain embodiments, AAV is genetically modified. In certain embodiments, AAV is chemically modified.

[0237] In certain embodiments, AAV has been genetically modified to produce AAV vectors with altered receptor usage, antigenicity, transduction efficiency and / or tissue tropism for gene therapy applications. In certain embodiments, AAV can be genetically modified to insert peptide ligands, antibodies, antibody fragments, MHC and / or receptors into the viral capsid. In certain embodiments, AAV can be genetically modified to insert peptides for labeling the viral capsid. Non-limiting examples of possible modifications can be found in Büning H., Bolyard CM, Hallek M., Bartlett JS, (2012) Modification and labeling of AAV vector particles, included in: Snyder R., Moullier P., eds., Adeno-associated viruses, Methods in Molecular Biology (Methods and Protocols), Vol. 807, Humana Press, which is incorporated herein in its entirety for all intended purposes.

[0238] In some embodiments, AAV has been chemically modified to change the tissue tropism of the AAV vector. For example, a chemically selective reaction that can be directed to a specific amino acid side chain can be used to change the charge, polarity, hydrophobicity, and hydrogen bonding potential within the receptor binding domain on the AAV capsid. The ability to change the composition of specific receptors on the surface of the AAV capsid allows the production of synthetic vectors with altered tissue tropism. In some embodiments, the chemically modified AAV vector may exhibit altered receptor usage, antigenicity, transduction efficiency, and / or tissue tropism of the chemically modified AAV vector. Non-limiting examples of possible modifications can be found in Büning H., Bolyard CM, Hallek M., Bartlett JS, (2012) Modification and labeling of AAV vector particles, included in: Snyder R., Moullier P., eds., adeno-associated virus, molecular biology methods (methods and protocols), Vol. 807, Humana Press, which is incorporated herein as a whole for all intended purposes.

[0239] In some embodiments, the AAV fraction represents the AAV fraction produced by the transfected host cells. In some embodiments, the AAV fraction represents the supernatant harvested from a cell culture (comprising host cells transfected with a three-plasmid system), wherein one plasmid of the system comprises a target gene or cDNA, and one plasmid encodes capsid protein VP1, capsid protein VP2 and / or capsid protein VP3. In some embodiments, VP1, VP2 and / or VP3 are AAV8 VP1, AAV8 VP2 and / or AAV8VP3. In some embodiments, VP1, VP2 and / or VP3 are AAV9 VP1, AAV9 VP2 and / or AAV9 VP3. Three-plasmid transfection for producing rAAV is known in the art. See, e.g., Qu et al., 2015 (supra); Mizukami et al., "AAV vector production and purification scheme" (doctoral dissertation), Department of Genetic Therapeutics, Center for Molecular Medicine, 1998; and Kotin et al., Hum Mol Genet 20 (R1): R2-R6 (2011). In some embodiments, transfection can be performed using an inorganic compound (e.g., calcium phosphate) or an organic compound, polyethyleneimine (PEI) or a non-chemical method (e.g., electroporation). In some embodiments, the host cell is an adherent cell. In some embodiments, the host cell is a suspension cell. In some embodiments, the host cell is a HEK293 cell or an Sf9 cell. In some embodiments, the cell culture comprises a serum-free and protein-free culture medium. In some embodiments, the chemical composition of the culture medium is defined and does not contain animal-derived components (e.g., hydrolysates). In some embodiments, the fraction containing rAAV particles represents a fraction containing HEK293 cells transfected with a three-plasmid system. In certain embodiments, the fractions comprising rAAV particles are described in U.S. Provisional Application No. 62 / 417,775 and WO2018128688, which are incorporated herein by reference for all intended purposes.

[0240] After culturing host cells (e.g., HEK293 cells) to produce AAV particles (e.g., AAV8, AAV9, etc.) and concentrating and / or filtering the clarified cell-free culture supernatant, the viral particles are loaded onto an affinity matrix. In certain embodiments, the viral particles are loaded into a solution having a pH of about 7.4 to about 7.8. In certain embodiments, the viral particles are loaded into a solution having a pH of about 8.3 to about 8.7. In certain embodiments, the viral particles are loaded into a solution having a pH of about 8.5. In certain embodiments, the pH is 8.3 to 8.7, and the solution comprises NaCl and TrisHCl. In certain embodiments, the viral particles are loaded into a solution comprising about 20 mM TrisHCl and about 150 mM NaCl and a pH of about 8.5.

[0241] Additional Steps and Considerations

[0242] The method of the present disclosure includes any combination of steps disclosed herein, and can be optionally combined with more than one additional step. Therefore, in exemplary aspects, the method of the present disclosure also includes the step of transfecting host cells with the three-plasmid system described herein. In exemplary aspects, the method of the present disclosure includes harvesting supernatant from a cell culture, and the cell culture comprises a host cell (e.g., HEK293 cell) transfected with the three-plasmid system. In exemplary aspects, the transfection and harvesting steps occur before the ultracentrifugation step described herein.

[0243] The methods of the present disclosure may include other additional steps that can further improve the purity of the AAV and remove other unwanted components and / or concentrate and / or adjust the fraction for subsequent steps.

[0244] In certain embodiments, an optional re-equilibration step is performed prior to the wash steps listed above.

[0245] In certain embodiments, prior to affinity purification of the AAV-containing solution from the host cell product, a pre-purification may be performed to remove one or more complex acidic protein structures and host cell DNA. Pre-purification may be performed by anion exchange in a flow through mode. A pre-purification step may be performed prior to any of the affinity purification methods described herein. One or more of the following can be removed by pre-purifying such an AAV-containing solution: histones (e.g., type 1 histone H2A, type 1-B histone H2B, histone H4, histone H1.4), 60S ribosomal proteins (e.g., 60S ribosomal protein L27, 60S ribosomal protein L6, and 60S ribosomal protein L30), cytoplasmic actin (e.g., cytoplasmic actin 1), tubulin (e.g., tubulin β-2A chain), heterogeneous nuclear ribonucleoprotein C, Rep68 protein, 37 kDa form of HEK293 laminin receptor (LamR 37 kDa), and ATP-dependent molecular chaperone HSC82.

[0246] In certain embodiments, the additional step may be an ultracentrifugation step.

[0247] In an exemplary aspect, the method includes a deep filtration step. In an exemplary aspect, the method includes: using a filtration device comprising cellulose and perlite and having a minimum permeability of about 500 L / m 2In an exemplary aspect, the method further comprises: using a filter with a minimum pore size of about 0.2 μm. In an exemplary aspect, after the depth filtration, filtering is performed through a filter with a minimum pore size of about 0.2 μm. In an exemplary aspect, one or both of the depth filter and the filter with a minimum pore size of about 0.2 μm are washed, and the wash solution is collected. In an exemplary aspect, the wash solution is combined together and combined with the filtrate obtained during the depth filtration and the filtration with the filter with a minimum pore size of about 0.2 μm.

[0248] In some embodiments, the method further comprises: contacting the AAV-containing solution with an anion exchanger prior to loading the AAV-containing solution onto the affinity resin, and eluting the AAV-containing solution from the anion exchanger. The anion exchanger can be operated in flow-through mode.

[0249] In exemplary aspects, the methods of the present disclosure include more than one chromatography step. In exemplary aspects, the methods include a negative chromatography step such that unwanted components bind to the chromatography resin and the desired AAV does not bind to the chromatography resin. In exemplary aspects, the methods include a negative anion exchange (AEX) chromatography step or an AEX chromatography step in a "non-binding mode". Advantages of the "non-binding mode" include relative ease of performing the procedure and conducting subsequent analysis.

[0250] Thus, in an exemplary embodiment, a method for purifying AAV particles comprises: subjecting the fraction containing the AAV particles to negative anion exchange (AEX) chromatography by adding the fraction to an AEX chromatography column or membrane under conditions that allow AAV to flow out of the AEX chromatography column or membrane; and collecting the AAV particles. In an exemplary aspect, the fraction is added to an AEX chromatography column or membrane having a loading buffer comprising about 100 mM to about 150 mM salt (e.g., NaCl), optionally, the pH of the loading buffer is about 8 to about 9. In an exemplary aspect, the loading buffer comprises about 115 mM to about 130 mM salt (e.g., NaCl), optionally, the loading buffer comprises about 120 mM to about 125 mM salt (e.g., NaCl). In an exemplary aspect, the negative AEX step is performed prior to the ultracentrifugation step described herein.

[0251] In exemplary aspects, the methods of the present disclosure include concentrating the AAV fraction using an ultrafiltration / diafiltration system. In exemplary aspects, the methods of the present disclosure include one or more tangential flow filtration (TFF) steps. In exemplary aspects, the AAV fraction undergoes ultrafiltration / diafiltration. In exemplary aspects, the AAV fraction is concentrated using an ultrafiltration / diafiltration system before performing a step comprising negative AEX chromatography, after performing a step comprising negative AEX chromatography, or before and after performing a step comprising negative AEX chromatography. In exemplary aspects, the TFF step is performed before the ultracentrifugation step described herein.

[0252] For example, enveloped virus inactivation may be particularly important when using the Baculo transfection system.In exemplary aspects, the disclosed methods include filtering a fraction comprising rAAV particles to remove viruses in the fraction that are larger in size than the rAAV particles.

[0253] Without wishing to be bound by theory, ethylene glycol by itself or in combination with another additive can inactivate such lipid enveloped viruses. Exemplary additives include nonionic detergents, aliphatic agents (e.g., TnBP), and detergents (e.g., polysorbates (e.g., Tween), Triton X100, TnBP). For example, the solvent-detergent mixture can include 1% Triton X100, 0.3% tri-n-butyl phosphate, and 0.3% Tween 80.

[0254] As summarized in Table 1 below, inactivation of lipid enveloped viruses "on-column" can be tested in various affinity chromatography runs.

[0255] Table 1: Solvent Detergent Treatments Used in Variants A, B and C

[0256]

[0257] The buffer wash X buffer containing DMSO can effectively trigger the elution of AAV9 but not AAV8 on CaptureSelect AAV8 resin at a near neutral pH (e.g., pH 8.0), which is surprising. The buffer wash X buffer containing DMSO can effectively trigger the elution of various AAVs, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV, BAAV, AAV (VR-195) and AAV (VR-355) on CaptureSelect AAVx resin at a near neutral pH (e.g., pH 8.0), which is surprising. In some embodiments, the AAV is an AAV5 serotype. In some embodiments, the AAV is an AAV6 serotype. In some embodiments, the AAV is an AAV8 serotype. In some embodiments, the AAV is an AAV9 serotype.

[0258] Without wishing to be bound by theory, Wash X Buffer is expected to have the activity of washing the chromatography column and / or inactivating or decomposing lipid enveloped viruses. It was not expected that Wash X Buffer would differentially elute AAV8 and AAV9.

[0259] In exemplary aspects, the method of the present disclosure includes one or more quality control steps, for example, a step of measuring the titer or specific activity of the AAV fraction obtained at one or more steps (e.g., after each step) of the process. In exemplary aspects, the method of the present disclosure includes an ELISA specific for AAV. In exemplary aspects, the ELISA is a sandwich ELISA. In exemplary aspects, the sandwich ELISA comprises antibodies specific for AAV epitopes. In exemplary aspects, the AAV epitope is a conformational epitope present on the assembled AAV capsid. As discussed herein, ELISA can replace qPCR as a way to determine the titer of the AAV fraction. In exemplary aspects, the method of the present disclosure includes detecting the AAV fraction by an AAV-specific ELISA, which does not include a method for detecting the titer by quantitative PCR. In exemplary aspects, the AAV-specific ELISA is sufficient to provide a representative reading of the titer of the AAV fraction because most of the capsids in the AAV fraction are complete capsids.

[0260] In exemplary aspects, the methods of the present disclosure include performing an ELISA specific for AAV after one or more steps of the present disclosure. In exemplary aspects, the methods of the present disclosure include detecting the AAV fraction obtained after depth filtration by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the methods of the present disclosure include detecting the AAV fraction obtained after concentrating the AAV fraction using an ultrafiltration / diafiltration system by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the methods of the present disclosure include detecting the AAV fraction obtained after the tangential flow filtration (TFF) step by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the methods of the present disclosure include detecting the AAV fraction obtained after negative anion exchange (AEX) chromatography by an AAV-specific ELISA to determine the specific activity of AAV in the fraction. In exemplary aspects, the methods of the present disclosure include detecting the AAV fraction obtained after the refining step by an AAV-specific ELISA to determine the specific activity of AAV in the fraction.

[0261] In some embodiments, the method further comprises: contacting the solution containing AAV with a filter, wherein the filter comprises positively charged groups, wherein the positively charged groups are effective to deplete acidic charged contaminants in the solution containing AAV. In some embodiments, the method further comprises: nanofiltration of the AAV fraction to remove viruses larger than 35 nm. In some embodiments, the method further comprises a refining step, wherein the refining step comprises performing cation exchange chromatography. Exemplary media for cation exchange chromatography include, but are not limited to, Capto TM S. S and In some embodiments, the method further comprises: detecting the AAV fraction by an AAV-specific (eg, AAV8 or AAV9-specific) ELISA. The AAV-specific ELISA may be a sandwich ELISA specific for AAV (eg, AAV8 or AAV9).

[0262] AAV Products

[0263] In another aspect, an AAV product produced by any of the methods described herein is provided.

[0264] Also provided herein are AAV products produced by the methods of the present disclosure. In exemplary aspects, the AAV product comprises at least about 10 μL of starting material (e.g., cell culture) produced from about 1000 μL of starting material (e.g., cell culture). 12 Virus particles (vp) or at least about 10 produced from about 1000 L of starting material (e.g., cell culture) 13Virus particles (vp). In exemplary aspects, the AAV product is an empty capsid produced by transfection of rep-cap and Ad helper plasmids in the absence of a transgenic plasmid. The purified empty plasmid can be used to deplete or remove antibodies specific for AAV antigens from the patient's blood.

[0265] In exemplary aspects, the AAV products of the present disclosure are highly pure, highly titer and suitable for clinical use in humans. In exemplary aspects, the AAV products include homogenous populations and highly pure AAV particles. In exemplary aspects, the AAV products include full-length vector DNA. In exemplary embodiments, the AAV products are substantially free of unwanted contaminants, including, but not limited to, AAV particles containing truncated or incomplete vector DNA, AAV particles with incomplete protein composition and oligomeric structure, or contaminating viruses (e.g., non-AAV, lipid enveloped viruses). In exemplary embodiments, the AAV products include a large amount of cDNA encoding target proteins. In exemplary aspects, the AAV products of the present disclosure are suitable for administration to humans. In exemplary aspects, the AAV products are sterile and / or good manufacturing practice (GMP) grade. In exemplary aspects, the AAV products meet the requirements of Chapter 1046 of the United States Pharmacopoeia or the European Pharmacopoeia for gene therapy drug products or the provisions of the U.S. Food and Drug Administration (USFDA) or the European Medicines Agency (EMA). In exemplary aspects, the AAV product is a ready-to-use product for direct administration to humans with little processing or handling.

[0266] In connection with the methods of the present invention, the AAV fraction is, in exemplary aspects, a concentrated AAV fraction. In certain embodiments, the AAV fraction comprises at least 1×10 10 pcs, 1×10 11 or 1×10 12 AAV capsids / mL. In certain embodiments, the AAV fraction contains at least 1×10 12 AAV capsids / mL. The AAV capsids may include empty AAV capsids and complete AAV capsids. In certain embodiments, the AAV fraction contains more complete AAV capsids than empty AAV and / or overfilled AAV capsids.

[0267] In certain embodiments, at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the AAV capsids eluted from the elution step are intact AAV capsids.

[0268] In certain embodiments, the methods for producing and purifying AAV described herein result in a product that contains at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% more intact capsids than a product obtained by a comparable procedure without the same washing regimen and elution at a lower temperature. , 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In certain embodiments, the methods for producing and purifying AAV described herein result in a product that contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains at least about 6% more intact capsids than a product obtained by a comparable procedure (without the same washing regimen and elution at a lower temperature). In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains at least about 10% more intact capsids than a product obtained by a comparable procedure (without the same washing regimen and elution at a lower temperature). In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains at least about 20% more intact capsids than a product obtained by a comparable procedure (without the same washing protocol and elution at a lower temperature). In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains about 6% more intact capsids than a product obtained by a comparable procedure (without the same washing protocol and elution at a lower temperature). In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains about 10% more intact capsids than a product obtained by a comparable procedure (without the same washing protocol and elution at a lower temperature).In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains about 16% more intact capsids than a product obtained by a comparable procedure (without the same washing protocol and elution at a lower temperature). In certain embodiments, the methods of producing and purifying AAV described herein result in a product that contains about 20% more intact capsids than a product obtained by a comparable procedure (without the same washing protocol and elution at a lower temperature).

[0269] In various embodiments, the yield (weight / volume) of AAV (e.g., AAV9) as measured by ITR-qPCR following the purification steps described herein is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% higher than the yield obtained by a comparable procedure without a wash step.

[0270] In various embodiments, the yield (weight / weight) of AAV (e.g., AAV9) as measured by ITR-qPCR following the purification steps described herein is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% higher than the yield obtained by a comparable procedure without a wash step.

[0271] Advantageously, the method can be extended to a large amount of starting material (e.g., cell culture). In certain embodiments, the method provided herein is a large-scale method capable of purifying AAV by a volume of at least or about 500L, at least or about 600L, at least or about 700L, at least or about 800L, at least or about 900L, at least or about 1000L. In certain embodiments, the method can be extended to a minimum volume of starting material (e.g., cell culture) of at least or about 1250L, at least or about 1500L, at least or about 2000L, at least or about 2500L, at least or about 3000L, at least or about 4000L, at least or about 5000L, at least or about 6000L, at least or about 7000L, at least or about 8000L, at least or about 9000L, at least or about 10,000L or more. For example, the method is performed with a minimum volume of about 1000 L, or about 10,000 L, or 25,000 L or more of the AAV-producing cell culture.

[0272] The methods of producing and purifying AAV described herein are also advantageous because they result in the production of high titer AAV. 10 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 11An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 12 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 13 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 14 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 15 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 16 An AAV product containing at least about 10 viral particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 17 An AAV product containing at least about 2×10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 16 An AAV product containing at least about 5×10 virus particles (vp) is produced from about 1000 L of starting material (e.g., cell culture). 17 An AAV product of 10 viral particles (vp) is produced from approximately 1000 L of starting material (e.g., cell culture).

[0273] Another advantage of the methods described herein is that the methods produce highly pure AAV products. In certain embodiments, the AAV products produced by the methods disclosed herein are substantially free of one or more contaminants: host cell proteins, host cell nucleic acids (e.g., free host cell DNA and free plasmid DNA), plasmid DNA, empty viral capsids, heat shock protein 70 (HSP70), lactate dehydrogenase (LDH), proteasomes, contaminating non-AAV viruses (e.g., lipid enveloped viruses), host cell culture components (e.g., antibiotics), mycoplasmas, pyrogens, bacterial endotoxins, cell debris (e.g., fragments composed of membrane lipids, proteins, and other biopolymers), and exogenous factors. When AAV is purified according to the methods for producing and purifying AAV described herein, one or more or even all of the following impurities may not be detected: histone H2A type 1, histone H2B type 1-B, histone H4, heat shock protein 70kDa 1A, pyruvate kinase PKM, elongation factor 2, ATP-citrate synthase, histone H1.4, immunoglobulin heavy chain constant region gamma 1 (immobilized ligand from acid elution method), 60S ribosomal protein L27, fructose-bisphosphate aldolase A, heat shock cognate protein 71 kDa, cytoplasmic actin 1, S-formylglutathione hydrolase, asparagine synthetase (glutamine hydrolysis), L-lactate dehydrogenase B chain, tubulin beta-2A chain, X-chromosome RNA binding motif protein, 60S ribosomal protein L6, cytoplasmic threonine tRNA ligase, immunoglobulin kappa constant, 60S ribosomal protein L30, WD repeat-containing protein 1, adenosylhomocysteine ​​enzyme, heterogeneous nuclear ribonucleoprotein C, protein Rep68, Thimet oligopeptidase, D-3-phosphoglycerate dehydrogenase, and ATP-dependent molecular chaperone HSC82. According to the methods for producing and purifying anionic AAV described herein, adding an anion exchange step prior to the washing step can also render the following substances undetectable: histone H1.4, 60S ribosomal protein L27, cytoplasmic actin 1, tubulin β-2A chain, 60S ribosomal protein L6, 60S ribosomal protein L30, heterogeneous nuclear ribonucleoprotein C, protein Rep68, and ATP-dependent molecular chaperone HSC82.

[0274] In an exemplary embodiment, the methods of the present disclosure provide a purified AAV product, wherein at least or about 50% of the contaminants found in the starting material (e.g., cell culture) are removed. In an exemplary embodiment, the methods of the present disclosure provide a purified AAV product, wherein at least or about 60% of the contaminants found in the starting material (e.g., cell culture) are removed. In an exemplary embodiment, the methods of the present disclosure provide a purified AAV product, wherein at least or about 70% of the contaminants found in the starting material (e.g., cell culture) are removed. In an exemplary embodiment, the methods of the present disclosure provide a purified AAV product, wherein at least or about 80% of the contaminants found in the starting material (e.g., cell culture) are removed. In an exemplary embodiment, the methods of the present disclosure provide a purified AAV product, wherein at least or about 90% of the contaminants found in the starting material (e.g., cell culture) are removed.

[0275] In certain embodiments, the methods of producing and purifying AAV described herein reduce the amount of impurities, including protein impurities, such as host cell (HC) impurities, by more than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.6%, 96.7%, 96.8%, 96.9%, 97.1%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1%, 97.9%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1%, 97. %, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%. In some embodiments, the impurity level of the AAV obtained from the elution step is ≤ 99.9%. In some embodiments, the impurity level of the AAV obtained from the elution step is ≤ 99.0%.

[0276] In certain embodiments, the methods for producing and purifying AAV described herein result in a product that is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 96.10%, 96.11%, 96.12%, 96.13%, 96.14%, 96.15%, 96.16%, 96.17%, 96.18%, 96.19%, 96.20%, 96.21%, 96.22%, 96.23%, 96.24%, 96.25%, 96.26%, 96.27%, 96.28%, 96.29%, 96.30%, 96.31%, 96.32%, 96.33%, 96.34%, 96.35%, 96.36%, 96.37%, 96.38%, 96.39%, 96.40%, 96.50%, 96.51%, 96.52%, 96.53%, 96.54%, 96.55%, 96.56%, 96.57%, 96.58%, 96.59%, 96.60%, 96.610%, 96.67%, 96.68%, 96.69%, 96.70%, 96.71%, 96.72%, 96.73%, 96 In some embodiments, the AAV obtained from the elution step has a purity level of 99.9% or more. In some embodiments, the AAV obtained from the elution step has a purity level of 99.0% or more.

[0277] In certain embodiments, the methods for producing and purifying AAV described herein result in a product that is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.6%, 96.7%, 96.8%, 96.9%, 97.1%, 97.2%, 97.3%, 97.4%, 97.6%, 97.7%, 97.8%, 97.9%, 97.1%, 97.9%, 97.1%, 97.2%, 97.3%, 97.9%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 97.9%, 97.1%, 97.9%, 97.1%, 97. In some embodiments, the purity level of the AAV obtained from the elution step is greater than 99.9% compared to the product obtained by a comparable procedure (not performing the same wash protocol and eluting at a lower temperature). In some embodiments, the AAV obtained from the elution step has a purity level greater than 99.0% compared to a product obtained by a comparable procedure that does not perform the same wash protocol and elutes at a lower temperature.

[0278] In certain embodiments, the AAV products produced by the methods of the present disclosure are suitable for administration to humans. In certain embodiments, the AAV is a recombinant AAV (rAAV). In certain embodiments, the AAV products produced by the methods of the present disclosure are sterile and / or of good manufacturing practice (GMP) grade. In certain embodiments, the AAV products produced by the methods of the present disclosure meet the requirements of Chapter 1046 of the United States Pharmacopoeia or the European Pharmacopoeia for gene therapy drug products or the regulations of the United States Food and Drug Administration (USFDA) or the European Medicines Agency (EMA).

[0279] In addition, the AAV products produced by the methods described herein are highly titered. The titer of an AAV product (e.g., an AAV8 or AAV9 product) can be described by: (1) in vivo bio-titer (e.g., production of active protein in mice), expressed as U (FIX or FVIII) / mL mouse plasma; or (2) in vitro bio-titer. In vitro bio-titer tests detect the potential of AAV vectors to infect cells (e.g., HepG2 cells), which express the target protein and secrete the target protein into the culture medium, and the quantity is determined by ELISA technology and / or enzyme activity. Suitable methods for measuring in vivo and in vitro bio-titer are known in the art and are also described herein.

[0280] In further embodiments, the AAV products produced by the methods described herein exhibit excellent specific activity. The "specific activity" of AAV is represented by the ratio of qPCR to μg AAV8. In an exemplary embodiment, the AAV products produced by the methods described herein show excellent GOI ratios per μg AAV, indicating that the AAV products have a large number of "complete" viral particles. In certain embodiments, the methods of the present disclosure include detecting AAV fractions by AAV-specific ELISA. In certain embodiments, the AAV-specific ELISA is sufficient to provide a representative reading of the titer of the AAV fraction because most of the capsids in the AAV fraction are complete capsids.

[0281] Non-limiting embodiments:

[0282] 1. A method for purifying an adeno-associated virus (AAV), wherein the method comprises:

[0283] (a) loading a solution containing AAV onto an affinity resin for AAV at room temperature under conditions that allow the AAV in the solution to bind to the affinity resin;

[0284] (b) performing at least one washing step at room temperature; and

[0285] (c) AAV was eluted from the affinity resin at a temperature below 18°C.

[0286] 2. The method according to embodiment 1, wherein the temperature in step (c) is 1°C to 12°C.

[0287] 3. The method according to embodiment 1, wherein the temperature in step (c) is 2°C to 8°C.

[0288] 4. The method of any one of embodiments 1 to 3, further comprising: contacting the AAV-containing solution with an anion exchanger before loading the AAV-containing solution onto the affinity resin, and eluting the AAV-containing solution from the anion exchanger.

[0289] 5. The method of any one of embodiments 1 to 4, wherein at least one of the washing steps comprises adding a buffer comprising an organic solvent and / or a detergent to the affinity resin.

[0290] 6. The method of embodiment 5, wherein the buffer comprises sodium acetate.

[0291] 7. The method of embodiment 5, wherein the buffer comprises TrisHCl and salt.

[0292] 8. The method according to embodiment 5, wherein the buffer comprises one or more of histidine, histidine-HCl, arginine-HCl, lysine-HCl, glycine, taurine, MES-Na, Bis-Tris and N-acetyl-D,L-tryptophan.

[0293] 9. The method according to embodiment 7 or 8, wherein the salt is NaCl.

[0294] 10. The method of embodiment 5, wherein the buffer comprises magnesium chloride.

[0295] 11. The method of embodiment 5, wherein the buffer comprises TrisHCl and ethylene glycol.

[0296] 12. The method of embodiment 5, wherein the buffer comprises arginine-HCl and one of sucrose and glycerol.

[0297] 13. The method of embodiment 5, wherein the buffer comprises taurine and ethylene glycol.

[0298] 14. The method of embodiment 5, wherein the buffer comprises arginine-HCl, lysine-HCl, and histidine-HCl.

[0299] 15. The method of embodiment 5, wherein the buffer comprises TrisHCl and DMSO.

[0300] 16. The method according to embodiment 5, wherein the organic solvent or detergent is polysorbate 80, ethylene glycol, sorbitol, mannitol, xylitol, DMSO, sucrose or trehalose.

[0301] 17. The method according to embodiment 5, wherein the detergent comprises one or more of Triton X100, polysorbate 80 and tri(n-butyl) phosphate (TNBP).

[0302] 18. The method of embodiment 17, wherein the detergent comprises polysorbate 80.

[0303] 19. The method of any one of embodiments 5 to 18, wherein the buffer comprises about 0.05% to about 30% (w / w) of an organic solvent or detergent.

[0304] 20. The method of embodiment 5, wherein the buffer comprises about 0.05% to about 0.2% (w / w) of an organic solvent or detergent.

[0305] 21. The method of any one of embodiments 1 to 20, wherein at least two washing steps are performed at room temperature.

[0306] 22. The method of any one of embodiments 1 to 21, wherein at least 3 washing steps are performed at room temperature.

[0307] 23. The method of any one of embodiments 1 to 22, wherein at least 4 washing steps are performed at room temperature.

[0308] 24. The method of embodiment 21, wherein two washing steps are performed.

[0309] 25. The method of embodiment 22, wherein three washing steps are performed.

[0310] 26. A method according to embodiment 23, wherein 4 washing steps are performed.

[0311] 27. The method of any one of embodiments 1 to 26, wherein the washing step is performed continuously.

[0312] 28. The method of any one of embodiments 1 to 27, wherein at least one wash buffer comprises about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt.

[0313] 29. A method according to embodiment 28, wherein at least one wash buffer comprises about 25 mM to about 100 mM TrisHCl and about 75 mM to about 250 mM salt.

[0314] 30. The method of embodiment 29, wherein at least one wash buffer comprises about 40 mM to about 60 mM TrisHCl and about 100 mM to about 150 mM salt.

[0315] 31. The method of any one of embodiments 28 to 30, wherein the pH of the wash buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, or about 8.0 to about 8.8.

[0316] 32. A method according to embodiment 31, wherein at least one wash buffer comprises about 50 mM TrisHCl and about 125 mM salt, and has a pH of about 8.5.

[0317] 33. The method of any one of embodiments 1 to 32, wherein at least one wash buffer comprises about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80.

[0318] 34. The method of embodiment 33, wherein at least one wash buffer comprises about 50 mM to about 200 mM sodium acetate and about 0.005% to about 0.3% (w / w) polysorbate 80.

[0319] 35. The method of embodiment 34, wherein at least one wash buffer comprises about 90 mM to about 110 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80.

[0320] 36. The method of any one of embodiments 33 to 35, wherein the pH of the wash buffer is about 5.0 to about 7.4, about 5.5 to about 7.0, or about 5.5 to about 6.5.

[0321] 37. The method of embodiment 36, wherein at least one wash buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0322] 38. The method of any one of embodiments 1 to 37, wherein at least one wash buffer comprises about 10 mM to about 200 mM TrisHCl and about 10% to about 75% (w / w) ethylene glycol.

[0323] 39. A method according to embodiment 38, wherein at least one wash buffer comprises about 25 mM to about 100 mM TrisHCl and about 25% to about 70% (w / w) ethylene glycol.

[0324] 40. The method of embodiment 39, wherein at least one wash buffer comprises about 40 mM to about 60 mM TrisHCl and about 40% to about 60% (w / w) ethylene glycol.

[0325] 41. The method of any one of embodiments 38 to 40, wherein the pH of the wash buffer is about 7.5 to about 9.2, about 8.0 to about 9.0, or about 8.0 to about 8.8.

[0326] 42. The method of claim 41, wherein at least one wash buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol at a pH of about 8.5.

[0327] 43. A method according to any one of embodiments 1 to 42, wherein at least one wash buffer comprises about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% (w / w) trehalose and about 0.0005% to about 1% (w / w) polysorbate 80.

[0328] 44. A method according to embodiment 43, wherein at least one wash buffer comprises about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% (w / w) trehalose and about 0.001% to about 0.1% (w / w) polysorbate 80.

[0329] 45. A method according to embodiment 44, wherein at least one wash buffer comprises about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% (w / w) trehalose and about 0.001% to about 0.05% (w / w) polysorbate 80.

[0330] 46. ​​The method of any one of embodiments 43 to 45, wherein the pH of the wash buffer is about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.4.

[0331] 47. A method according to embodiment 46, wherein at least one wash buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose and about 0.005% (w / w) polysorbate 80, and has a pH of about 7.0.

[0332] 48. A method according to any one of embodiments 1 to 47, wherein at least one wash buffer comprises about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80.

[0333] 49. A method according to embodiment 48, wherein at least one wash buffer comprises about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80.

[0334] 50. A method according to embodiment 49, wherein at least one wash buffer comprises about 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate 80.

[0335] 51. The method of any one of embodiments 48 to 50, wherein the pH of the wash buffer is about 6.0 to about 8.8, about 6.5 to about 8.5, or about 7.0 to about 8.0.

[0336] 52. A method according to embodiment 51, wherein at least one wash buffer comprises about 20 mM TrisHCl, about 150 mM salt and about 0.1% (w / w) polysorbate 80, and has a pH of about 7.4.

[0337] 53. A method according to any one of embodiments 1 to 52, wherein step (c) comprises eluting the AAV with at least one elution buffer.

[0338] 54. A method according to embodiment 53, wherein at least one elution buffer is the same as at least one wash buffer.

[0339] 55. A method according to embodiment 54, wherein at least one elution buffer is the same as the last wash buffer used in the last wash step before eluting AAV in step (c).

[0340] 56. A method according to embodiment 54, wherein the first elution buffer is the same as the last wash buffer used in the last wash step before eluting AAV in step (c).

[0341] 57. A method according to any one of embodiments 53 to 56, wherein at least one elution buffer comprises about 10 mM to about 200 mM glycine, about 1 mM to about 100 mM histidine, about 20 mM to about 500 mM salt, about 1% to about 10% (w / w) trehalose and about 0.0005% to about 1% (w / w) polysorbate 80.

[0342] 58. A method according to embodiment 57, wherein at least one elution buffer comprises about 30 mM to about 80 mM glycine, about 5 mM to about 20 mM histidine, about 50 mM to about 200 mM salt, about 3% to about 8% trehalose and about 0.001% to about 0.1% (w / w) polysorbate 80.

[0343] 59. A method according to embodiment 58, wherein at least one elution buffer comprises about 40 mM to about 60 mM glycine, about 5 mM to about 15 mM histidine, about 90 mM to about 110 mM salt, about 4% to about 6% (w / w) trehalose and about 0.001% to about 0.05% (w / w) polysorbate 80.

[0344] 60. The method of any one of embodiments 57 to 59, wherein the pH of the wash buffer is about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.0 to about 7.4.

[0345] 61. A method according to embodiment 60, wherein at least one elution buffer comprises about 50 mM glycine, about 10 mM histidine, about 100 mM salt, about 5% (w / w) trehalose and about 0.005% (w / w) polysorbate 80, and has a pH of about 7.0.

[0346] 62. A method according to any one of embodiments 53 to 61, wherein at least one wash buffer comprises about 1 mM to about 200 mM TrisHCl, about 50 mM to about 500 mM salt, and about 0.001% to about 1% (w / w) polysorbate 80.

[0347] 63. A method according to embodiment 62, wherein at least one wash buffer comprises about 5 mM to about 50 mM TrisHCl, about 75 mM to about 250 mM salt, and about 0.005% to about 0.3% (w / w) polysorbate 80.

[0348] 64. A method according to embodiment 63, wherein at least one wash buffer comprises about 10 mM to about 30 mM TrisHCl, about 140 mM to about 160 mM salt, and about 0.05% to about 0.2% (w / w) polysorbate 80.

[0349] 65. The method of any one of embodiments 62 to 64, wherein the pH of the wash buffer is about 6.0 to about 8.8, about 6.5 to about 8.5, or about 7.0 to about 8.0.

[0350] 66. A method according to embodiment 65, wherein at least one wash buffer comprises about 20 mM TrisHCl, about 150 mM salt and about 0.1% (w / w) polysorbate 80, and has a pH of about 7.4.

[0351] 67. The method of any one of embodiments 1 to 66, wherein the first wash step and the third wash step comprise adding a buffer comprising about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt at a pH of about 7.5 to about 9.2 to the affinity resin; and

[0352] The second wash step comprises adding a buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80 at a pH of about 5.0 to about 7.4 to the affinity resin.

[0353] 68. The method of any one of embodiments 1 to 66, wherein the first wash step and the third wash step comprise adding a buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80 at a pH of about 5.0 to about 7.4 to the affinity resin; and

[0354] The second wash step comprises adding a buffer comprising about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt at a pH of about 7.5 to about 9.2 to the affinity resin.

[0355] 69. The method of any one of embodiments 1 to 66, wherein the first wash step and the third wash step comprise adding a buffer comprising about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt at a pH of about 7.5 to about 9.2 to the affinity resin; and

[0356] The second and fourth wash steps include adding a buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80 at a pH of about 5.0 to about 7.4 to the affinity resin.

[0357] 70. The method of any one of embodiments 1 to 66, wherein the first wash step and the third wash step comprise adding a buffer comprising about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80 at a pH of about 5.0 to about 7.4 to the affinity resin; and

[0358] The second and fourth wash steps include adding a buffer comprising about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt at a pH of about 7.5 to about 9.2 to the affinity resin.

[0359] 71. A method according to embodiment 67 or 69, wherein the first buffer and the third buffer contain about 50 mM TrisHCl and about 125 mM salt, and the pH is about 8.5.

[0360] 72. The method of embodiment 68 or 70, wherein the first buffer and the third buffer comprise about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0361] 73. The method of embodiment 67 or 69, wherein the second buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0362] 74. A method according to embodiment 68 or 70, wherein the second buffer comprises about 50 mM TrisHCl and about 125 mM salt at a pH of about 8.5.

[0363] 75. The method of embodiment 69, wherein the fourth buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0364] 76. A method according to embodiment 70, wherein the fourth buffer comprises about 50 mM TrisHCl and about 125 mM salt, and the pH is about 8.5.

[0365] 77. A method according to embodiment 67 or 70, wherein step (c) comprises adding a buffer to the affinity resin, wherein the buffer comprises about 10 mM to about 200 mM TrisHCl and about 50 mM to about 500 mM salt, and the pH is about 7.5 to about 9.2.

[0366] 78. A method according to embodiment 68 or 69, wherein step (c) comprises adding a buffer to the affinity resin, wherein the buffer comprises about 10 mM to about 2000 mM sodium acetate and about 0.001% to about 1% (w / w) polysorbate 80, and the pH is about 5.0 to about 7.4.

[0367] 79. A method according to embodiment 77, wherein the buffer comprises about 50 mM TrisHCl and about 125 mM salt, and the pH is about 8.5.

[0368] 80. The method of embodiment 78, wherein the buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80 at a pH of about 6.0.

[0369] 81. A method according to any one of embodiments 1 to 80, wherein at least one wash buffer and / or elution buffer comprises about 50 mM to about 500 mM of sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 mM to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO and tri(n-butyl) phosphate (TNBP), and the pH of at least one wash buffer and / or elution buffer is about 5.2 to about 6.8.

[0370] 82. A method according to any one of embodiments 1 to 81, wherein at least one wash buffer and / or elution buffer comprises about 30 mM to about 200 mM TrisHCl or arginine-HCl and about 75 mM to about 500 mM salt, and a pH of about 7.5 to about 9.2.

[0371] 83. A method according to any one of embodiments 1 to 82, wherein at least one wash buffer and / or elution buffer comprises about 20 mM to about 80 mM arginine-HCl and about 50 mM to about 200 mM salt, and a pH of about 7.3 to about 8.8.

[0372] 84. A method according to any one of embodiments 1 to 83, wherein at least one wash buffer and / or elution buffer comprises about 50 mM to about 200 mM taurine and 0.2% to 1.5% (w / w) PEG (e.g., PEG6000), and the pH is about 5.2 to about 6.8.

[0373] 85. A method according to any one of embodiments 1 to 84, wherein at least one and / or elution wash buffer comprises about 30 mM to about 300 mM glycine and a pH of about 7.5 to about 9.2.

[0374] 86. A method according to any one of embodiments 1 to 85, wherein at least one wash buffer and / or elution buffer comprises about 20 mM to about 150 mM taurine, about 30% to about 75% (w / w) ethylene glycol and 0.05% to 0.2% octylpyranoglucoside, and a pH of about 7.3 to about 8.8.

[0375] 87. A method according to any one of embodiments 1 to 86, wherein at least one wash buffer and / or elution buffer comprises about 80 mM to about 400 mM Bis-Tris and about 10 grams to about 20 grams of a solvent / detergent mixture, wherein the solvent / detergent mixture comprises Triton-X 100, polysorbate 80, and TNBP in a ratio of about 11:3:3 (by weight), and the pH of the at least one wash buffer and / or elution buffer is about 5.2 to about 6.8.

[0376] 88. A method according to any one of embodiments 1 to 87, wherein at least one wash buffer and / or elution buffer comprises about 5 mM to about 20 mM sodium citrate and a pH of about 7.5 to about 9.2.

[0377] 89. A method according to any one of embodiments 1 to 88, wherein at least one wash buffer and / or elution buffer comprises about 50 mM to about 200 mM arginine-HCl, about 50 mM to about 200 mM lysine-HCl, about 50 mM to about 200 mM histidine-HCl, about 1 mM to about 4 mM N-acetyl-D,L-tryptophan and about 10% to about 40% (w / w) polysorbate 80, and a pH of about 7.3 to about 8.8.

[0378] 90. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 2000 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, the first buffer having a pH of about 5.2 to about 6.8;

[0379] A second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 30 mM to about 200 mM TrisHCl and about 75 mM to about 500 mM salt, the second buffer having a pH of about 7.5 to about 9.2; and

[0380] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 30 mM to about 200 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 7.3 to about 8.8.

[0381] 91. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 500 mM sodium salt of 2-(N-morpholino)ethanesulfonic acid (MES-Na), about 3 mM to about 30 mM EDTA, and a solvent / detergent mixture comprising polysorbate 80, DMSO, and tri(n-butyl)phosphate (TNBP), the first buffer having a pH of about 5.2 to about 6.8;

[0382] A second washing step comprises adding a second buffer to the affinity resin, wherein the second buffer comprises about 30 mM to about 200 mM TrisHCl or arginine-HCl and about 75 mM to about 500 mM salt, the second buffer having a pH of about 7.5 to about 9.2; and

[0383] The third washing step comprises adding a third buffer to the affinity resin, the third buffer comprising about 20 mM to about 80 mM arginine-HCl and about 50 mM to about 200 mM salt, the third buffer having a pH of about 7.3 to about 8.8.

[0384] 92. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM taurine and 0.2% to 1.5% (w / w) PEG (e.g., PEG 6000), the first buffer having a pH of about 5.2 to about 6.8;

[0385] A second wash step comprises adding a second buffer to the affinity resin, the second buffer comprising about 30 mM to about 300 mM glycine, the second buffer having a pH of about 7.5 to about 9.2; and

[0386] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 20 mM to about 150 mM taurine, about 30% to about 75% (w / w) ethylene glycol, and 0.05% to 0.2% (w / w) octylpyranoglucoside, and the pH of the third buffer is about 7.3 to about 8.8.

[0387] 93. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 80 mM to about 400 mM Bis-Tris and about 10 grams to about 20 grams of a solvent / detergent mixture comprising Triton-X 100, polysorbate 80, and TNBP in a ratio of about 11:3:3 (by weight), the first buffer having a pH of about 5.2 to about 6.8;

[0388] A second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 5 mM to about 20 mM sodium citrate, the second buffer having a pH of about 7.5 to about 9.2; and

[0389] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 50 mM to about 200 mM arginine-HCl, about 50 mM to about 200 mM lysine-HCl, about 50 mM to about 200 mM histidine-HCl, about 1 mM to about 4 mM N-acetyl-D,L-tryptophan and about 10% to about 40% (w / w) polysorbate 80, and the pH of the third buffer is about 7.3 to about 8.8.

[0390] 94. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, the first buffer having a pH of about 5.2 to about 6.8;

[0391] A second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 20 nM to about 100 mM TrisHCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8; and

[0392] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 20 mM to 100 mM TrisHCl, about 40% to about 60% (w / w) ethylene glycol, and the pH of the third buffer is about 7.5 to about 8.8.

[0393] 95. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 nM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, the first buffer having a pH of about 5.2 to about 6.8;

[0394] A second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 20 nM to about 100 mM TrisHCl and about 50 nM to about 200 nM salt, the second buffer having a pH of about 7.5 to about 8.8; and

[0395] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 20 mM to 100 mM TrisHCl, about 40% to about 60% (w / w) ethylene glycol, and the pH of the third buffer is about 7.5 to about 8.8.

[0396] 96. The method of any one of embodiments 7 or 29 to 95, wherein the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, sodium acetate, and a combination of more than one of NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl, and sodium acetate.

[0397] 97. The method of embodiment 96, wherein the salt is NaCl.

[0398] 98. The method of any one of embodiments 7 or 29 to 97, wherein the concentration of the salt does not exceed 500 mM.

[0399] 99. The method of any one of embodiments 7 or 29 to 97, wherein the concentration of the salt does not exceed 200 mM.

[0400] 100. The method of any one of embodiments 28 to 32 or 67 to 95, wherein the buffer comprises about 50 mM TrisHCl and about 125 mM NaCl at a pH of about 8.5.

[0401] 101. The method of any one of embodiments 1 to 66, wherein the first wash step comprises adding a first buffer to the affinity resin, the first buffer comprising about 50 mM to about 200 mM sodium acetate and about 0.05% to about 0.2% (w / w) polysorbate 80, the first buffer having a pH of about 5.5 to about 6.5;

[0402] A second washing step comprises adding a second buffer to the affinity resin, the second buffer comprising about 10 mM to about 70 mM TrisHCl and about 75 mM to about 250 mM NaCl, the second buffer having a pH of about 8.0 to about 9.0; and

[0403] The third washing step comprises adding a third buffer to the affinity resin, wherein the third buffer comprises about 10 mM to about 70 mM TrisHCl and about 30% to about 75% (w / w) ethylene glycol, and the pH of the third buffer is about 8.0 to about 9.0.

[0404] 102. A method according to embodiment 101, wherein the method further comprises performing a fourth washing step before the first washing step, the fourth washing step comprising adding a fourth buffer to the affinity resin, the fourth buffer comprising about 10 mM to about 30 mM TrisHCl and about 75 mM to about 250 mM NaCl, and the pH of the fourth buffer is about 6.5 to about 8.0.

[0405] 103. The method of embodiment 100 or 102, wherein the first buffer comprises about 100 mM sodium acetate and about 0.1% (w / w) polysorbate 80, and the pH of the first buffer is about 6.0.

[0406] 104. The method of any one of embodiments 100 to 103, wherein the second buffer comprises about 50 mM TrisHCl and about 125 mM NaCl, and the pH of the second buffer is about 8.5.

[0407] 105. The method of any one of embodiments 100 to 104, wherein the third buffer comprises about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH of the third buffer is about 8.0.

[0408] 106. The method of any one of embodiments 1 to 105, wherein acidic components are removed.

[0409] 107. A method according to embodiment 106, wherein the acidic component is host cell DNA (e.g., HEK293 DNA) and the acidic component is reduced to a value below 250 pg / μg AAV antigen (measured by qPCR).

[0410] 108. A method according to embodiment 106, wherein the acidic component is host cell DNA (e.g., HEK293 DNA) and the acidic component is reduced to a value below 250 pg / μg AAV antigen (measured by ELISA).

[0411] 109. A method according to any one of embodiments 1 to 108, wherein the elution comprises continuously linearly increasing the conductivity of the elution buffer by gradient elution.

[0412] 110. The method of any one of embodiments 1 to 109, wherein the elution comprises continuously linearly increasing the concentration of the organic solvent by gradient elution.

[0413] 111. A method according to any one of embodiments 1 to 110, wherein the elution comprises contacting the affinity resin with an elution buffer comprising sodium acetate, glycine, histidine, NaCl and / or polysorbate 80.

[0414] 112. The method of embodiment 111, wherein the salt concentration is about 50 mM to 200 mM.

[0415] 113. The method of embodiment 111 or 112, wherein the pH is 5.5 to 9.0.

[0416] 114. The method of any one of embodiments 111 to 113, wherein the eluting comprises contacting the affinity resin with an elution buffer comprising 50 mM to 200 mM NaCl and 30 mM to 80 mM TrisHCl.

[0417] 115. A method according to embodiment 114, wherein the pH of the elution buffer is 8.0 to 9.0.

[0418] 116. A method according to embodiment 114, wherein the elution buffer contains at least about 55% (w / w) ethylene glycol.

[0419] 117. A method according to any one of embodiments 1 to 116, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising approximately 50 mM to 200 mM sodium acetate, 0.05% to 0.2% (w / w) polysorbate 80, and the pH of the elution buffer is approximately 5.5 to 6.5.

[0420] 118. A method according to any one of embodiments 1 to 117, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising 30mM to 80mM glycine, 5mM to 20mM histidine, 50mM to 200mM NaCl, 3% to 8% (w / w) trehalose and 0.001% to 0.1% (w / w) polysorbate 80, and the pH of the elution buffer is 6.5 to 7.5.

[0421] 119. The method of embodiment 118, wherein the eluting further comprises:

[0422] (a) contacting the affinity resin with a first elution buffer, the first elution buffer comprising 50 mM to 200 mM sodium acetate and 0.05% to 0.2% (w / w) polysorbate 80, the pH of the first elution buffer being about 5.5 to 6.5;

[0423] (b) contacting the affinity resin with a second elution buffer comprising 30 mM to 80 mM glycine, 5 mM to 20 mM histidine, 50 mM to 200 mM NaCl, 3% to 8% (w / w) trehalose, and 0.001% to 0.1% (w / w) polysorbate 80, wherein the pH of the elution buffer is 6.5 to 7.5; and

[0424] (c) contacting the affinity resin with a third elution buffer, wherein the third elution buffer comprises 50 mM to 200 mM NaCl and 30 mM to 80 mM TrisHCl, and the pH of the third elution buffer is 8.0 to 9.0.

[0425] 120. The method of any one of embodiments 1 to 119, wherein the steps are performed continuously.

[0426] 121. A method according to any one of embodiments 1 to 120, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising about 2 mM magnesium chloride, about 50 mM arginine-HCl, about 750 mM to about 1000 mM NaCl and at least about 50% (w / w) glycerol, and the pH of the elution buffer is at least about 8.0.

[0427] 122. A method according to any one of embodiments 1 to 121, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising about 2 mM magnesium chloride, about 50 mM taurine, about 600 mM to about 1000 mM NaCl, about 0.05 to about 0.2% (w / w) octylpyranoglucoside and about 60% (w / w) ethylene glycol, and the pH of the elution buffer is at least about 7.8.

[0428] 123. The method of embodiment 122, wherein the eluting further comprises:

[0429] (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising about 20 mM to about 100 mM TrisHCl and about 75 mM to about 250 mM NaCl, the fifth buffer having a pH of about 8.0 to about 8.8; and

[0430] (b) contacting the affinity resin with a second elution buffer comprising about 1 M ammonium sulfate, about 50 mM TrisHCl, and about 50% (w / w) ethylene glycol, the pH of the second elution buffer being at least about 6.8.

[0431] 124. A method according to embodiment 123, wherein the steps are performed continuously.

[0432] 125. A method according to any one of embodiments 1 to 124, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising about 1 M ammonium sulfate, about 50 mM TrisHCl and about 50% (w / w) ethylene glycol, and the pH of the elution buffer is at least about 6.8.

[0433] 126. A method according to any one of embodiments 1 to 125, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising about 20% (w / w) sucrose, about 10% (w / w) sorbitol, about 5% (w / w) mannitol or about 5% (w / w) sucrose, about 15% (w / w) glycerol, about 50 mM histidine and about 750 mM to about 1000 mM NaCl, and the pH of the elution buffer is at least about 7.8.

[0434] 127. The method of embodiment 126, wherein the eluting further comprises:

[0435] (a) contacting the affinity resin with a fifth buffer, the fifth buffer comprising about 20 mM to about 100 mM histidine and about 80 mM to about 120 mM NaCl, the fifth buffer having a pH of about 8.0 to about 8.8; and

[0436] (b) contacting the affinity resin with a second elution buffer comprising about 20 mM to about 100 mM histidine, about 600 mM to about 900 mM NaCl, and about 5% to 60% (w / w) DMSO, the fifth elution buffer having a pH of about 6.5 to about 8.5.

[0437] 128. A method according to embodiment 127, wherein the steps are performed continuously.

[0438] 129. A method according to any one of embodiments 1 to 128, wherein the elution comprises contacting the affinity resin with an elution buffer, the elution buffer comprising about 100 mM glycine-HCl and about 200 mM NaCl, and the pH of the elution buffer is about 2.5.

[0439] 130. The method of any one of embodiments 109 to 129, wherein the pH of the elution buffer is about 8.0.

[0440] 131. The method of any one of embodiments 109 to 129, wherein the pH of the elution buffer is 8.0.

[0441] 132. A method according to any one of embodiments 109 to 131, wherein the elution comprises gradually increasing the concentration of counter ions.

[0442] 133. A method according to any one of embodiments 109 to 132, wherein the elution comprises gradually increasing the concentration of the organic solvent.

[0443] 134. A method according to any one of embodiments 1 to 133, wherein the salt in the elution buffer is selected from monovalent anions, divalent anions or polyvalent anions, such as chloride, acetate, sulfate and citrate.

[0444] 135. A method according to any one of embodiments 1 to 134, wherein the method further comprises: eluting by using a gradient of 0 to 100% (w / w) 20mM to 50mM hydrochloric acid / 800mM to 1200mM NaCl in 0.5mM to 2.0mM hydrochloric acid.

[0445] 136. A method according to any one of embodiments 1 to 135, wherein the HC impurity level of the AAV obtained from the elution step is ≤99.9%.

[0446] 137. A method according to any one of embodiments 1 to 136, wherein the HC impurity level of the AAV obtained from the elution step is ≤99.0%.

[0447] 138. The method of any one of claims 1 to 137, wherein the purity level of the AAV obtained from the elution step is greater than 99.0%.

[0448] 139. The method of any one of claims 1 to 138, wherein the purity level of the AAV obtained from the elution step is greater than 99.9%.

[0449] 140. The method of any one of embodiments 1 to 139, wherein at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% of the AAV capsids eluted by elution step (c) are intact AAV capsids.

[0450] 141. The method of any one of embodiments 1 to 140, wherein the affinity resin is an AAVx resin.

[0451] 142. The method of any one of embodiments 1 to 141, wherein the AAV is AAV9.

[0452] 143. A method according to embodiment 142, wherein the AAV9 comprises a peptide, and the peptide comprises the sequence of SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3.

[0453] 144. A method according to any one of embodiments 1 to 143, wherein the method further comprises: contacting the solution containing AAV with a filter, wherein the filter comprises positively charged groups, and the positively charged groups can effectively deplete acidic charged contaminants in the solution containing AAV.

[0454] 145. The method of any one of embodiments 1 to 144, further comprising: nanofiltration of the AAV fraction to remove viruses larger than 35 nm.

[0455] 146. The method of any one of embodiments 1 to 145, further comprising a refining step comprising performing cation exchange chromatography.

[0456] 147. A method according to any one of embodiments 1 to 146, wherein the method further comprises: detecting the AAV fraction by AAV-specific ELISA.

[0457] 148. A method according to embodiment 147, wherein the AAV-specific ELISA is a sandwich ELISA specific for AAV.

[0458] 149. An AAV product produced by the method described in any one of embodiments 1 to 148.

[0459] Example

[0460] The following examples are given merely to illustrate the present invention and are not intended to limit its scope in any way.

[0461] Example 1

[0462] The following example describes an exemplary method for transfecting a HEK293 cell line using a three-plasmid system to produce rAAV particles containing nucleic acid encoding a protein of interest.

[0463] Adherent HEK293 cells are grown under suspension conditions in a commercially available culture medium that is chemically defined and free of animal-derived components, proteins, and serum, such as described in WO2018128688, paragraphs

[0146] -

[0150] , which are incorporated herein by reference for all intended purposes. The cells are transfected with three plasmids: (1) a helper plasmid that provides helper virus functions essential for proliferative AAV infection; (2) a repcap-plasmid that carries all information about the capsid production, replication, and packaging of the virus; and (3) a plasmid containing a gene of interest (GOI), which is packaged into the resulting rAAV particles. The size of the GOI is 2.6 kB to 3.0 kB. The rAAV particles carrying the gene of interest enter the HEK293 cell line within 3 to 5 days after transfection.

[0464] The supernatant of the transfected HEK293 cell culture is harvested, for example as described in paragraphs

[0151] -

[0155] , Table 1 and Table 2 of WO2018128688, which are incorporated herein by reference for all intended purposes. The harvested supernatant is concentrated and conditioned (diafiltered), for example as described in paragraphs

[0156] -

[0160] , Table 3 and Table 4 of WO2018128688, which are incorporated herein by reference for all intended purposes. The diafiltered concentrate is subjected to negative chromatography, for example as described in paragraphs

[0161] -

[0165] and Table 5 of WO2018128688, which are incorporated herein by reference for all intended purposes.

[0465] Example 2

[0466] AAV9 production was performed in the HEK293 cell line after transfection with a three-plasmid system (containing cDNA encoding the protein of interest and VP1, VP2 and VP3 of AAV9). AAV9 contains approximately 2.6 kB to 3.0 kB of vector DNA. The clarified cell-free culture supernatant was concentrated and diafiltered using Pall Omega T series components (100 kDa). The viral particles were loaded onto a membrane adsorber (Mustang Q; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5). The pH-adjusted load was obtained by adjusting the flow-through containing AAV9 to a pH of 7.4 to 7.8 with 25% HCl.

[0467] The following procedure was performed. First, the POROS-containing column was activated with 5× column volumes of a buffer solution (containing 100 mM glycine, 200 mM NaCl, pH 2.0). TM CaptureSelect TM AAVx affinity resin (Thermo Fisher, catalog number 36742) with an ID of 16 mm, a bed height of 50 ± 0.5 mm, and an area of ​​2.01 cm 2 and a chromatography column with a volume of approximately 10 mL. The chromatography column was then equilibrated with at least 5× column volumes of 50 mM TrisHCl, pH 8.5, and 125 mM NaCl. The pH adjusted sample was added to the column containing POROS TM CaptureSelect TM AAVx affinity resin on a chromatography column.

[0468] The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80 (i.e., polysorbate 80), pH 6.0) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). For all these wash steps, the linear flow rate was 60 cm / h.

[0469] Elution was performed using 5× column volumes of W2 above at a lower temperature of +2°C to +8°C. Then 5× column volumes of the following secondary elution buffer (ELT buffer) were added to the column at a temperature of +2°C to +8°C: 50 mM glycine, 10 mM histidine, 100 mM NaCl, 5% trehalose, 0.003% Crillet TM 4HP (i.e. polysorbate 80), pH 7.0. Then, 5× column volumes of the following elution buffer are added to the column at a temperature of +2°C to +8°C: 50mM TrisHCl and 125mM NaCl, pH 8.5. The linear flow rate for these elution steps is 5 cm / h. Then, 5× column volumes of purified water are added to the column at a temperature of +2°C to +8°C. A gradient elution is then performed. At a linear flow rate of 15 cm / h and a temperature of +2 to +26°C, 15× column volumes of a gradient of 1 mM to 20 mM HCl (in purified water) are added to empty the column.

[0470] The above procedure is described in more detail in Table 2, where "CV" represents the number of column volumes of solution added in that step.

[0471] Table 2

[0472]

[0473] The method of cooling the column from room temperature to about +2°C to +8°C comprises:

[0474] - Place the chromatography slide The Pure 150 is placed in a cooling cabinet, the Unichromat 1500;

[0475] - Equip the chromatographic column with a temperature jacket (water cooling);

[0476] - Use cold buffer for elution; or

[0477] - A combination of the above methods.

[0478] The samples collected were each analyzed by ITR-qPCR, ELISA for AAV antigens, and ELISA for HEK293 HCP to assess the yield and whether losses may have occurred during these steps.

[0479] ELISA was used to measure the amount of AAV9 antigen. ELISA was performed on a TECAN Roboter system using an AAV-9 titration ELISA kit (Cat. No. PRAAV9, Progen, Heidelberg, Germany). Briefly, a monoclonal antibody specific for the AAV9 capsid (AAV8 / 9 antibody ("ADK8 / 9 antibody", Catalog No. 03-651161, American Research Products, Inc., Waltham, Massachusetts)) was coated on a microtiter strip and used to capture AAV9 particles from the AAV fraction. The captured AAV9 particles were detected in two steps. In the first step, a biotin-conjugated monoclonal antibody specific for the ADK8 / 9 antibody was bound to the immune complex (of ADK8 / 9 and ADK8 / 9 antibodies). A streptavidin peroxidase conjugate was added to the immune complex bound to the biotin-conjugated monoclonal antibody, and the streptavidin peroxidase conjugate reacted with biotin. Peroxidase substrate solution is added and a color reaction occurs (proportional to the amount of bound AAV particles). The color reaction is measured photometrically at 450 nm.

[0480] The ITR-qPCR assay is used to determine the genome copy titer by quantifying the inverted tandem repeats found in the vector encoding the gene of interest (e.g., human factor VIII or human factor IX). HEK-HCP is a measure of residual host cell protein measured by ELISA. LDH is determined by a colorimetric activity assay.

[0481] In the in vitro biopotency assay, the viral vector AAV9 infects a hepatic target cell line, which subsequently secretes a functional, measurable encoded protein into the culture medium. In the first step, HepG2 target cells are transduced and infected with AAV9. During incubation, the encoded protein is released into the cell supernatant. In the second step, the activity of the encoded protein that enters the cell culture supernatant is measured directly by an activity assay. The measured values ​​of the AAV9 samples are given as a percentage relative to the reference material. This method allows for a quantitative assessment of the biological function of the AAV9 gene therapy vector.

[0482] SDS-PAGE analysis was performed to determine whether there was a reduction in heat shock protein 70 kDa (HSP70) when the test procedure with a washing step was used instead of the comparison procedure. Western Blot was performed as follows: anti-Hsp70 antibody (Abcam, catalog number ab79852) was used as the primary antibody (1:2000 dilution) for 2 hours and goat anti-rabbit IgG (H+L) AP conjugate (Sigma, catalog number A8025) was used as the secondary antibody (1:1000 dilution) for 1 hour.

[0483] SDS-PAGE silver staining assays were performed to determine the overall level of impurities present. Analytical ultracentrifugation (AUC) was performed to quantify the amount of AAV9 present and to determine the relative amounts of intact capsids, empty capsids, and capsids with extra DNA compared to intact capsids (ie, overfilled).

[0484] Western Blot was performed using 12% anti-AAV antibody to determine the level and purity of AAV9 recovered after purification according to the test procedure and the comparative procedure. Western Blot was performed using monoclonal antibodies against VP1, VP2 and VP3 of AAV9 as primary antibodies and goat anti-mouse ALP antibody (Sigma, catalog number A4656) as secondary antibody.

[0485] These assays are summarized in Table 3 below.

[0486] Table 3

[0487]

[0488] Example 3

[0489] AAV9 production was performed in the HEK293 cell line after transfection with a three-plasmid system (containing cDNA encoding the protein of interest and VP1, VP2 and VP3 of AAV9). AAV9 contains approximately 2.6 kB to 3.0 kB of vector DNA. The clarified cell-free culture supernatant was concentrated and diafiltered using Pall Omega T series components (100 kDa). The viral particles were loaded onto a membrane adsorber (Mustang Q; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5). The pH-adjusted load was obtained by adjusting the flow-through containing AAV9 to a pH of 8.2 to 8.7 with 25% HCl.

[0490] The following procedure was performed. Note: All buffers disclosed in this example were prepared at room temperature and the pH of all buffers was measured at room temperature. First, the POROS-containing precipitate was activated with 5× column volumes of 100 mM glycine and 200 mM NaCl (pH 2.0). TM CaptureSelect TM AAVx affinity resin (Thermo Fisher, catalog number 36742) with an ID of 16 mm, a bed height of 50 ± 0.5 mm, and an area of ​​2.01 cm 2 and a column with a volume of approximately 10 mL. The column was then equilibrated with at least 10× column volumes of 50 mM TrisHCl, pH 8.5, and 125 mM NaCl. The pH adjusted sample was added to the column containing POROSTM CaptureSelect TM AAVx affinity resin on a chromatography column.

[0491] The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). For all these wash steps, the linear flow rate was 60 cm / h.

[0492] Elution was performed by adding 10× column volumes of W1 as above, but at a lower temperature of +2°C to +8°C. For elution (i.e., step 7 in Table 4 below), the chromatography carriage, chromatography column, and buffer were cooled to below +8°C by placing everything in a cooling cabinet (Unichromat 1500). The linear flow rate for elution was 5 cm / h. The column was then stripped with 5× column volumes of 100 mM glycine and 200 mM NaCl (pH 2.0).

[0493] The above procedure is described in more detail in Table 4, where "CV" represents the number of column volumes of solution added in that step.

[0494] Table 4

[0495]

[0496] Figure 1 The chromatogram for the above procedure is shown. The results of the protocol of Table 4 are shown in Table 5 below. The E1 pool "cold elution" value reflects the amount after step 7 of Table 4 above. The "stripping" value reflects the amount after step 9 of Table 4 above. Table 5 clearly shows that elution is caused by the temperature shift to +2 to +8°C, because W1 and W3 (which do not result in significant elution of AAV9) use the same buffer (i.e. 125mM NaCl and 50mM TrisHCl, pH 8.5) but are performed at room temperature (i.e. +18 to 25°C).

[0497] Table 5

[0498]

[0499] LOQ: below the detection limit; -: cannot be calculated (because the amount of DNA is below the detection limit); E1 combined solution is the eluent.

[0500] In addition, the measured impurity content of the method described in Table 4 is shown in Table 6 below. The impurity sample was collected for the initial sample and again after step 7.

[0501] Table 6

[0502]

[0503] Table 7 shows the % intact capsid and % overfilled capsid (determined based on particle size). As used herein, particle size is given in Svedbergs scale (based on the sedimentation rate of the particles).

[0504] Table 7

[0505]

[0506] As used herein, the term "qPCR yield" refers to the percentage of qPCR present compared to the initial amount of qPCR in the sample. As used herein, the term "antigenic yield" refers to the percentage of AAV9 present compared to the initial amount of AAV9 in the sample. The yield measured by qPCR was 75.8% (Table 5) and AAV9 with intact capsids was 87.5% (Table 7), indicating that the low temperature elution protocol described in this example provides a substantial enrichment of AAV9 intact capsids.

[0507] The chromatographic data from the above procedure were compared to a standard elution procedure (100 mM glycine and 200 mM NaCl, pH 2.7 as shown in Table 8) and the results are shown below in Table 9. The starting material was the same for both runs.

[0508] Table 8

[0509]

[0510] Table 9

[0511]

[0512] Tables 10 and 11 provide additional data for the standard elution program of Table 8.

[0513] Table 10

[0514]

[0515] Table 11

[0516]

[0517] As shown in Table 5, AAV9 binds to CaptureSelect TMAAVx resin, it was surprisingly and unexpectedly found that the bound AAV9 could then be eluted in the same buffer system by a temperature change from above +18°C to below +8°C. Therefore, the temperature change protocol has the benefit of gentle elution at low temperatures, helping to maintain the structure and infectivity of the AAV particles. Likewise, the use of a mild elution buffer in a manufacturing environment is also easy to perform and more efficient because the elution step does not require a buffer change. In addition, it was advantageously found that the intact AAV9 capsids were eluted by lowering the temperature, while the empty, non-specific AAV9 vectors remained bound until they were eluted by the acidic elution solution of the stripping step (see Table 7).

[0518] Example 4

[0519] Example 3 demonstrates that the use of a temperature change from above +18°C to below +8°C, using a buffer of 125 mM NaCl and 50 mM TrisHCl (pH 8.5), can be used to extract the TM Elution of AAV9 from AAVx. This example was performed to investigate the possibility of eluting AAV9 from AAVx using an alternative buffer system using a temperature change from above +18°C to below +8°C. After loading AAV9 onto the AAVx resin, a buffer at the higher temperature range was first added as a wash buffer, followed by a buffer at the lower temperature as an elution buffer.

[0520] AAV9 production was performed in the HEK293 cell line after transfection with a three-plasmid system (containing cDNA encoding the protein of interest and VP1, VP2 and VP3 of AAV9). AAV9 contains approximately 2.6 kB to 3.0 kB of vector DNA. The clarified cell-free culture supernatant was concentrated and diafiltered using Pall Omega T series components (100 kDa). Viral particles were loaded onto a membrane adsorber (Mustang Q; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5). The pH-adjusted load was obtained by adjusting the flow-through containing AAV9 to a pH of 8.2 to 8.7 with 25% HCl.

[0521] The following procedure was performed. Note: All buffers disclosed in this example were prepared at room temperature and the pH of all buffers were measured at room temperature.

[0522] Three buffers with increasing pH and conductivity were first added at higher temperatures (+18 to +28°C) to confirm that AAV9 was still bound to the ligand. After the column was cooled to +2°C to +8°C (the chromatographic slide, column and buffer were all lowered to below +8°C by placing everything in a cooling cabinet (Unichromat 1500)), the same buffers were then added to study the elution characteristics. First, the POROS-containing column was activated with 5× column volumes of buffer (containing 100 mM glycine, 200 mM NaCl, pH 2.0). TM CaptureSelect TM AAVx affinity resin (Thermo Fisher, catalog number 36742) with an ID of 16 mm, a bed height of 50 ± 0.5 mm, and an area of ​​2.01 cm 2 and a column with a volume of approximately 10 mL. The column was then equilibrated with at least 5× column volumes of 50 mM TrisHCl, pH 8.5, and 125 mM NaCl. The pH adjusted sample was added to the column containing POROS TM CaptureSelect TM AAVx affinity resin on a chromatography column.

[0523] The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). For all these wash steps, the linear flow rate was 60 cm / h.

[0524] Elution was first performed by adding 5× column volumes of W2 as above, but at a lower temperature of +2°C to +8°C at a linear flow rate of 5 cm / h. Then 5× column volumes of the following secondary elution buffer were added to the column at a temperature of +2°C to +8°C: 50 mM glycine, 10 mM histidine, 100 mM NaCl, 5% trehalose, 0.005% Crillet 4HP, pH 7.0 (ELT-buffer). Then 5× column volumes of the following elution buffer were added to the column at a temperature of +2°C to +8°C: 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The linear flow rate for these elution steps was 30 cm / h. Then 5× column volumes of purified water were added to the column at a temperature of +2°C to +8°C. A gradient elution was then performed. A gradient of 1 mM to 20 mM HCl / 200 mM NaCl in purified water was added over 15 x column volumes at a linear flow rate of 20 cm / h and a temperature of +2°C to +8°C.

[0525] The column was washed by adding 5× column volumes of the following regeneration buffer: 20 mM HCl and 200 mM NaCl in purified water, followed by 5× column volumes of 50 mM TrisHCl and 125 mM NaCl (pH 8.5) at 60 cm / h, followed by 5× column volumes of 1 M L-arginine monohydrochloride + 200 mM NaCl at a linear flow rate of 30 cm / h and a temperature of +2°C to +8°C.

[0526] The above procedure is described in more detail in Table 12, where "CV" represents the number of column volumes of solution added in that step.

[0527] Table 12

[0528]

[0529]

[0530] The results of the above procedure (particularly the percent yield of AAV9 eluted from the column) are shown in Table 13 below. Figure 2A-2B The chromatograms of the above procedure are shown.

[0531] Table 13

[0532]

[0533] LOQ: below the limit of detection; --: cannot be calculated (because the amount of DNA is below the limit of detection); *: the combined solution of E1.1 and E1.2, the eluate was collected at the main peak of E1.1 and the tail of E1.2.

[0534] As shown, elution in a lower temperature range was also confirmed with buffers other than 125 mM NaCl and 50 mM TrisHCl (pH 8.5). In addition, the measured impurity levels during the process described in Table 12 are shown in Table 14 below. Impurity samples were collected after each elution step (steps 8a, 8b, 9, 10 and 12).

[0535] Table 14

[0536]

[0537] The comparative results of low temperature elution of the buffers of Example 3 and Example 4 are shown in Table 15 below.

[0538] Table 15

[0539]

[0540]

[0541] Table 16

[0542]

[0543] na = not applicable; * = not measured (because the concentration was too low for AUC analysis).

[0544] Elution was confirmed with 100 mM sodium acetate, 0.1% (w / w) polysorbate (pH 6.0) at +2°C to +8°C (rather than room temperature) (see Table 16). It is worth noting that the pH and conductivity of 100 mM sodium acetate at low temperature are opposite to those of 125 mM NaCl, 50 mM TrisHCl (pH 8.5), as elution efficiency generally increases with increasing pH from pH 6 to pH 8.5. Therefore, Example 4 demonstrates that temperature is the driving force for elution, and that pH and conductivity have less influence on elution than temperature. It was also demonstrated that this affinity method with temperature variation resulted in higher levels of intact AAV capsids.

[0545] Thus, Examples 3 and 4 demonstrate that the mode of action for elution of AAV9 from AAVx depends primarily on lowering the temperature and is independent of the buffer system.

[0546] Example 5

[0547] Batch adsorption of AAV9 was performed according to the following procedure. Two groups with the above two different resins were first incubated with a solution containing AAV9 at room temperature to bind AAV9, and then eluted at a lower temperature. The procedure is summarized in Table 17 below. Note: All buffers disclosed in this example were prepared at room temperature and the pH of all buffers were measured at room temperature.

[0548] The steps of Example 5 are as follows: Balance: Add 0.2 g of resin to a 15 mL centrifuge tube and wash with 10 mL of 125 mM NaCl, 50 mM TrisHCl (pH 8.5 ± 0.2). Centrifuge the suspension at 5500 RPM (HERAEUS MEGAFUGE 16R, Thermo Fisher Scientific) for 10 minutes and discard the supernatant. Add 9.6 g of the sample to the washed / equilibrated resin and incubate at room temperature for 15 to 16 hours. Centrifuge the suspension at 5500 RPM for 10 minutes. Divide the supernatant into equal parts and detect the AAV9 antigen. Resuspend the precipitate with cold 1 mL of 125 mM NaCl, 50 mM TrisHCl (+2°C to +8°C) at pH 8.5 ± 0.2 and incubate for 30 minutes. Centrifuge the suspension at 5500 RPM for 10 minutes. Divide the supernatant into equal parts and detect the AAV9 antigen. AAV9 contains approximately 2.6 kB to 3.0 kB of vector DNA. For elution, cold buffer was used and the experiment was performed in a cold room.

[0549] Table 17

[0550]

[0551] The results of the above procedure (particularly the percent yield of AAV9 eluted from the column) are shown in Table 18 below.

[0552] Table 18

[0553]

[0554] In addition, a second set used a different resin incubated at a lower temperature to investigate the binding properties under these conditions. This process is summarized in Table 19 below.

[0555] Table 19

[0556]

[0557] The results of the above procedure (particularly the percent yield of AAV9 eluted from the column) are shown in Table 20 below.

[0558] Table 20

[0559]

[0560] AAV9 is compatible with CaptureSelect TM AAV9 binding, elution cannot be triggered at lower temperatures. Therefore, this example shows that low temperature elution is a characteristic of the interaction between AAV9 and AAVx affinity resin.

[0561] It was also observed that AAV8 and AAV6 did not elute from the AAVx resin when the temperature was changed from above +18° C. to below +8° C. In contrast, AAV8 and AAV6 required more stringent conditions (e.g., see Example 6 below) to elute from the AAVx affinity resin.

[0562] Example 6

[0563] AAV8 was produced in the HEK293 cell line after transfection with a three-plasmid system (containing cDNA encoding the protein of interest and VP1, VP2 and VP3 of AAV8). The clarified cell-free culture supernatant was concentrated and diafiltered using Pall Omega T series modules (100 kDa). Viral particles were loaded onto a membrane adsorber (Mustang Q; Pall part number XT140MSTGQP05) under non-binding conditions (i.e., in a solution containing 125 mM NaCl and 50 mM TrisHCl at pH 8.5). The pH-adjusted load was obtained by adjusting the flow-through containing AAV8 to a pH of 8.3 to 8.7 using 25% HCl.

[0564] The following test procedure was performed. Note: All buffers disclosed in this example were prepared at room temperature and the pH of all buffers was measured at room temperature. First, the POROS-containing 5× column volume buffer (containing 100 mM glycine, 200 mM NaCl, pH 2.0) was activated. TM CaptureSelect TM AAVx affinity resin (Thermo Fisher, catalog number 36742) with an ID of 16 mm, a bed height of 50 ± 0.5 mm, and an area of ​​2.01 cm 2 and a chromatography column with a volume of approximately 10 mL. The chromatography column was then equilibrated with at least 5× column volumes of 50 mM TrisHCl, pH 8.5, and 125 mM NaCl. The pH adjusted sample was added to the column containing POROS TM CaptureSelect TM AAVx affinity resin on a chromatography column.

[0565] The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). The column was then washed with 5× column volumes of wash 2 (W2: 100 mM sodium acetate and 0.1% Tween 80, pH 6.0) at room temperature (18°C to 26°C). For all these wash steps, the linear flow rate was 60 cm / h.

[0566] For elution, the chromatography carriage, column and buffer were cooled to below +8°C by placing everything in a cooling cabinet (Unichromat 1500). Elution was first performed by adding 5× column volumes of W2 as above, but at a lower temperature of +2°C to +8°C with a linear flow rate of 5 cm / h. Then, at a temperature of +2°C to +8°C, 5× column volumes of the following secondary elution buffer were added to the chromatography column: 50 mM glycine, 10 mM histidine, 100 mM NaCl, 5% trehalose, 0.005% Crillet 4HP, pH 7.0 (ELT-buffer). Then, at a temperature of +2°C to +8°C, 5× column volumes of the following elution buffer were added to the chromatography column: 50 mM TrisHCl and 125 mM NaCl, pH 8.5. The linear flow rate for these elution steps was 30 cm / h. Then, at a temperature of +2°C to +8°C, 5× column volumes of purified water were added to the chromatography column. Gradient elution was then performed. A gradient of 1 mM to 20 mM HCl / 200 mM NaCl (in purified water) was added over 15 x column volumes at a linear flow rate of 20 cm / h and a temperature of +2°C to +8°C.

[0567] The above procedure is described in more detail in Table 21, where "CV" represents the number of column volumes of solution added in that step.

[0568] Table 21

[0569]

[0570]

[0571] Table 22 below and Figure 3 The results of the above procedure (particularly the percentage yield of AAV8 eluted from the column) are shown in .

[0572] Table 22

[0573]

[0574] As shown, AAV8 could not be eluted from AAVx by shifting the temperature from +2°C to +8°C.

[0575] Example 7

[0576] The following procedure is an example of a single wash protocol. First, the POROS-containing column was activated with 5× column volumes of buffer (containing 100 mM glycine, 200 mM NaCl, pH 2.0). TM CaptureSelect TM AAVx affinity resin (Thermo Fisher, catalog number 36742) with an ID of 16 mm, a bed height of 50 ± 0.5 mm, and an area of ​​2.01 cm 2 The column was then equilibrated with 10× column volumes of 50 mM TrisHCl and 125 mM NaCl (pH 8.5). The pH adjusted sample was added to the column containing POROS TM CaptureSelect TM AAVx affinity resin on a chromatography column.

[0577] The column was then washed with 5× column volumes of Wash 1 (W1: 50 mM TrisHCl and 125 mM NaCl, pH 8.5) at room temperature (18°C to 26°C). Elution was performed by using 5× to 10× column volumes of W1 as above, but at a lower temperature of +2 to +8°C. The column was then stripped.

[0578] The above procedure is described in more detail in Table 23, where "CV" represents the number of column volumes of solution added in that step.

[0579] Table 23

[0580]

[0581] Example 8

[0582] The following sequence shows an example of an AAV9 VP1 sequence according to some embodiments of the present disclosure (SEQ ID NO: 1).

[0583]

[0584]

[0585] The following sequence shows an example of an AAV9 VP2 sequence according to some embodiments of the present disclosure, wherein the AAV9 VP2 sequence comprises the sequence of SEQ ID NO: 2.

[0586]

[0587]

[0588] The following sequence shows an example of an AAV9 VP2 sequence according to some embodiments of the present disclosure, wherein the AAV9 VP3 sequence comprises the sequence of SEQ ID NO: 3.

[0589]

[0590] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Claims

1. A method for purifying adeno-associated virus (AAV), wherein: The method comprises: (a) loading a solution containing AAV onto an affinity resin for AAV at room temperature under conditions that allow the AAV in the solution to bind to the affinity resin; (b) performing at least one washing step at room temperature; and (c) eluting the AAV from the affinity resin at a temperature of 1° C. to 12° C.; Wherein, the AAV is AAV9, and the affinity resin is POROS TM CaptureSelect TM AAVx resin.

2. The method according to claim 1, wherein: The temperature in step (c) is 2°C to 8°C.

3. The method according to claim 1 or 2, wherein: The method further includes contacting the AAV-containing solution with an anion exchanger prior to loading the AAV-containing solution onto the affinity resin, and eluting the AAV-containing solution from the anion exchanger.

4. The method according to any one of claims 1 to 3, wherein: Perform at least 2 washing steps at room temperature.

5. The method according to any one of claims 1 to 4, wherein: Perform at least 3 washing steps at room temperature.

6. The method according to any one of claims 1 to 5, wherein: Perform at least 4 wash steps at room temperature.

7. The method according to claim 4, wherein: Two washing steps were performed.

8. The method according to claim 5, wherein: Perform 3 washing steps.

9. The method according to claim 6, wherein: Four washing steps were performed.

10. The method according to any one of claims 4 to 9, wherein: The washing steps are performed continuously.

11. The method according to any one of claims 1 to 10, wherein: At least one wash buffer comprises 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt.

12. The method according to claim 11, wherein: The at least one wash buffer comprises 25 mM to 100 mM TrisHCl and 75 mM to 250 mM salt.

13. The method according to claim 12, wherein: The at least one wash buffer comprises 40 mM to 60 mM TrisHCl and 100 mM to 150 mM salt.

14. The method according to any one of claims 11 to 13, wherein: The pH of the at least one wash buffer is between 7.5 and 9.

2.

15. The method according to any one of claims 11 to 13, wherein: The pH of the at least one wash buffer is between 8.0 and 9.

0.

16. The method according to any one of claims 11 to 13, wherein: The pH of the at least one wash buffer is between 8.0 and 8.

8.

17. The method according to any one of claims 14 to 16, wherein: The at least one wash buffer comprises 50 mM TrisHCl and 125 mM salt at a pH of 8.

5.

18. The method according to any one of claims 1 to 17, wherein: At least one wash buffer comprises 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80.

19. The method according to claim 18, wherein: The at least one wash buffer comprises 50 mM to 200 mM sodium acetate and 0.005% to 0.3% w / w polysorbate 80.

20. The method according to claim 19, wherein: The at least one wash buffer comprises 90 mM to 110 mM sodium acetate and 0.05% to 0.2% w / w polysorbate 80.

21. The method according to any one of claims 18 to 20, wherein: The pH of the at least one wash buffer is between 5.0 and 7.

4.

22. The method according to any one of claims 18 to 20, wherein: The pH of the at least one wash buffer is between 5.5 and 7.

0.

23. The method according to any one of claims 18 to 20, wherein: The pH of the at least one wash buffer is between 5.5 and 6.

5.

24. The method according to any one of claims 21 to 23, wherein: The at least one wash buffer comprises 100 mM sodium acetate and 0.1% w / w polysorbate 80 at a pH of 6.

0.

25. The method according to any one of claims 1 to 24, wherein: At least one wash buffer comprises 10 mM to 200 mM TrisHCl and 10% to 75% w / w ethylene glycol.

26. The method according to claim 25, wherein: The at least one wash buffer comprises 25 mM to 100 mM TrisHCl and 25% to 70% w / w ethylene glycol.

27. The method according to claim 26, wherein: The at least one wash buffer comprises 40 mM to 60 mM TrisHCl and 40% to 60% w / w ethylene glycol.

28. A method according to any one of claims 25 to 27, wherein: The pH of the at least one wash buffer is between 7.5 and 9.

2.

29. The method according to any one of claims 25 to 27, wherein: The pH of the at least one wash buffer is between 8.0 and 9.

0.

30. The method according to any one of claims 25 to 27, wherein: The pH of the at least one wash buffer is between 8.0 and 8.

8.

31. The method according to any one of claims 28 to 30, wherein: The at least one wash buffer comprises 50 mM TrisHCl and 50% w / w ethylene glycol at a pH of 8.

5.

32. The method according to any one of claims 1 to 31, wherein: At least one wash buffer comprises 10 mM to 200 mM glycine, 1 mM to 100 mM histidine, 20 mM to 500 mM salt, 1% to 10% w / w trehalose, and 0.0005% to 1% w / w polysorbate 80.

33. The method of claim 32, wherein: The at least one wash buffer comprises 30 mM to 80 mM glycine, 5 mM to 20 mM histidine, 50 mM to 200 mM salt, 3% to 8% w / w trehalose, and 0.001% to 0.1% w / w polysorbate 80.

34. The method of claim 33, wherein: The at least one wash buffer comprises 40 mM to 60 mM glycine, 5 mM to 15 mM histidine, 90 mM to 110 mM salt, 4% to 6% w / w trehalose, and 0.001% to 0.05% w / w polysorbate 80.

35. The method according to any one of claims 32 to 34, wherein: The pH of the at least one wash buffer is between 6.0 and 8.

0.

36. The method according to any one of claims 32 to 34, wherein: The pH of the at least one wash buffer is between 6.5 and 7.

5.

37. The method according to any one of claims 32 to 34, wherein: The pH of the at least one wash buffer is between 7.0 and 7.

4.

38. A method according to any one of claims 35 to 37, wherein: The at least one wash buffer comprises 50 mM glycine, 10 mM histidine, 100 mM salt, 5% w / w trehalose and 0.005% w / w polysorbate 80 at a pH of 7.

0.

39. The method according to any one of claims 1 to 38, wherein: At least one wash buffer comprises 1 mM to 200 mM TrisHCl, 50 mM to 500 mM salt, and 0.001% to 1% w / w polysorbate 80.

40. The method of claim 39, wherein: The at least one wash buffer comprises 5 mM to 50 mM TrisHCl, 75 mM to 250 mM salt, and 0.005% to 0.3% w / w polysorbate 80.

41. The method of claim 40, wherein: The at least one wash buffer comprises 10 mM to 30 mM TrisHCl, 140 mM to 160 mM salt, and 0.05% to 0.2% w / w polysorbate 80.

42. The method according to any one of claims 39 to 41, wherein: The pH of the at least one wash buffer is between 6.0 and 8.

8.

43. The method according to any one of claims 39 to 41, wherein: The pH of the at least one wash buffer is between 6.5 and 8.

5.

44. A method according to any one of claims 39 to 41, wherein: The pH of the at least one wash buffer is between 7.0 and 8.

0.

45. A method according to any one of claims 42 to 44, wherein: The at least one wash buffer comprises 20 mM TrisHCl, 150 mM salt and 0.1% w / w polysorbate 80 at a pH of 7.

4.

46. ​​The method according to any one of claims 1 to 45, wherein: Step (c) comprises eluting the AAV with at least one elution buffer.

47. The method of claim 46, wherein: At least one elution buffer is the same as at least one wash buffer.

48. The method of claim 47, wherein: At least one elution buffer is the same as the last wash buffer used in the last wash step prior to eluting the AAV in step (c).

49. The method of claim 47, wherein: The first elution buffer is the same as the final wash buffer used in the final wash step before eluting the AAV in step (c).

50. The method according to any one of claims 46 to 49, wherein: The at least one elution buffer comprises 10 mM to 200 mM glycine, 1 mM to 100 mM histidine, 20 mM to 500 mM salt, 1% to 10% w / w trehalose, and 0.0005% to 1% w / w polysorbate 80.

51. The method of claim 50, wherein: The at least one elution buffer comprises 30 mM to 80 mM glycine, 5 mM to 20 mM histidine, 50 mM to 200 mM salt, 3% to 8% trehalose, and 0.001% to 0.1% w / w polysorbate 80.

52. The method of claim 51, wherein: The at least one elution buffer comprises 40 mM to 60 mM glycine, 5 mM to 15 mM histidine, 90 mM to 110 mM salt, 4% to 6% w / w trehalose, and 0.001% to 0.05% w / w polysorbate 80.

53. The method according to any one of claims 50 to 52, wherein: The pH of the at least one elution buffer is between 6.0 and 8.

0.

54. The method according to any one of claims 50 to 52, wherein: The pH of the at least one elution buffer is between 6.5 and 7.

5.

55. The method according to any one of claims 50 to 52, wherein: The pH of the at least one elution buffer is between 7.0 and 7.

4.

56. A method according to any one of claims 53 to 55, wherein: The at least one elution buffer comprises 50 mM glycine, 10 mM histidine, 100 mM salt, 5% w / w trehalose and 0.005% w / w polysorbate 80 at a pH of 7.

0.

57. The method according to any one of claims 46 to 56, wherein: The at least one elution buffer comprises 1 mM to 200 mM TrisHCl, 50 mM to 500 mM salt, and 0.001% to 1% w / w polysorbate 80.

58. The method of claim 57, wherein: The at least one elution buffer comprises 5 mM to 50 mM TrisHCl, 75 mM to 250 mM salt, and 0.005% to 0.3% w / w polysorbate 80.

59. The method of claim 58, wherein: The at least one elution buffer comprises 10 mM to 30 mM TrisHCl, 140 mM to 160 mM salt, and 0.05% to 0.2% w / w polysorbate 80.

60. The method according to any one of claims 57 to 59, wherein: The pH of the at least one elution buffer is between 6.0 and 8.

8.

61. The method according to any one of claims 57 to 59, wherein: The pH of the at least one elution buffer is between 6.5 and 8.

5.

62. The method according to any one of claims 57 to 59, wherein: The pH of the at least one elution buffer is between 7.0 and 8.

0.

63. The method according to any one of claims 60 to 62, wherein: The at least one elution buffer comprises 20 mM TrisHCl, 150 mM salt and 0.1% w / w polysorbate 80 at a pH of 7.

4.

64. The method according to any one of claims 1 to 63, wherein: The first and third washing steps include adding a buffer comprising 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt at a pH of 7.5 to 9.2 to the affinity resin; as well as The second wash step comprises adding a buffer comprising 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80 at a pH of 5.0 to 7.4 to the affinity resin.

65. The method according to any one of claims 1 to 63, wherein: The first washing step and the third washing step include adding a buffer to the affinity resin, the buffer comprising 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80, with a pH of 5.0 to 7.4; as well as The second wash step involves adding a buffer comprising 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt at a pH of 7.5 to 9.2 to the affinity resin.

66. The method according to any one of claims 1 to 63, wherein: The first and third washing steps include adding a buffer comprising 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt at a pH of 7.5 to 9.2 to the affinity resin; as well as The second and fourth washing steps include adding a buffer comprising 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80 at a pH of 5.0 to 7.4 to the affinity resin.

67. The method according to any one of claims 1 to 63, wherein: The first washing step and the third washing step include adding a buffer to the affinity resin, the buffer comprising 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80, with a pH of 5.0 to 7.4; as well as The second and fourth washing steps include adding a buffer comprising 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt at a pH of 7.5 to 9.2 to the affinity resin.

68. The method of claim 64 or 66, wherein: The first buffer and the third buffer used in the first washing step and the third washing step, respectively, each contained 50 mM TrisHCl and 125 mM salt, and had a pH of 8.

5.

69. The method of claim 65 or 67, wherein: The first buffer and the third buffer used in the first washing step and the third washing step, respectively, each contained 100 mM sodium acetate and 0.1% w / w polysorbate 80, and had a pH of 6.

0.

70. The method of claim 64 or 66, wherein: The second buffer used in the second washing step contained 100 mM sodium acetate and 0.1% w / w polysorbate 80, pH 6.

0.

71. The method of claim 65 or 67, wherein: The second buffer used in the second wash step contained 50 mM TrisHCl and 125 mM salt, pH 8.

5.

72. The method of claim 66, wherein: The fourth buffer used in the fourth washing step contained 100 mM sodium acetate and 0.1% w / w polysorbate 80, with a pH of 6.

0.

73. The method of claim 67, wherein: The fourth buffer used in the fourth washing step contained 50 mM TrisHCl and 125 mM salt, with a pH of 8.

5.

74. The method of claim 64 or 67, wherein: Step (c) comprises adding a buffer to the affinity resin, wherein the buffer comprises 10 mM to 200 mM TrisHCl and 50 mM to 500 mM salt, and the pH is 7.5 to 9.

2.

75. The method of claim 65 or 66, wherein: Step (c) comprises adding a buffer to the affinity resin, wherein the buffer comprises 10 mM to 2000 mM sodium acetate and 0.001% to 1% w / w polysorbate 80, and the pH is 5.0 to 7.

4.

76. The method of claim 74, wherein: The buffer contained 50 mM TrisHCl and 125 mM salt, pH 8.

5.

77. The method of claim 75, wherein: The buffer contained 100 mM sodium acetate and 0.1% w / w polysorbate 80, with a pH of 6.

0.

78. The method of any one of claims 11 to 77, wherein: The salt is selected from: NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl or sodium acetate.

79. The method according to any one of claims 11 to 77, wherein: The salt is selected from the group consisting of: NaCl, KCl, MgCl2, CaCl2, sodium citrate, LiCl, CsCl or sodium acetate.

80. The method of claim 78, wherein: The salt is NaCl.

81. The method of any one of claims 11 to 17, 64 to 68, 71, 73 or 74, wherein The buffer contained 50 mM TrisHCl and 125 mM NaCl, pH 8.

5.

82. The method according to any one of claims 1 to 81, wherein The purity level of AAV obtained from the elution step was above 99.0%.

83. The method according to any one of claims 1 to 82, wherein: The purity level of AAV obtained from the elution step was above 99.9%.

84. The method according to any one of claims 1 to 83, wherein: At least 60% of the AAV capsids eluted by the elution step (c) are intact AAV capsids.

85. The method according to any one of claims 1 to 84, wherein: The AAV9 comprises a peptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2 and / or SEQ ID NO:3.

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