Virus isolation

Abstract

A method for separating smaller viruses from larger viruses from a fluid is provided, comprising the steps of: (a) passing a fluid containing the smaller viruses and the larger viruses through a porous filter comprising a porous medium at a differential pressure of at least 20 psid to obtain the smaller viruses; (b) reducing the pressure to 3 psid or less; and (c) passing additional fluid containing the viruses through the porous filter comprising the porous medium at a pressure of at least 20 psid to obtain additional smaller viruses.

Description

[Background technology]

[0001] Some processes for separating desired viruses from fluids exhibit reduced yields and / or reduced yields due to contamination with other unwanted viruses.

[0002] Improved methods are needed to improve the separation of desired viruses from virus-containing fluids. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent No. 4,340,479 [Patent Document 2] U.S. Patent No. 4,925,572 [Patent Document 3] U.S. Patent No. 7,318,800 [Patent Document 4] U.S. Patent No. 6,113,784 [Patent Document 5] U.S. Patent No. 5,152,905 [Patent Document 6] U.S. Patent No. 5,443,743 [Patent Document 7] U.S. Patent No. 5,472,621 [Patent Document 8] U.S. Patent No. 6,074,869 Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention seeks to ameliorate at least some of the shortcomings of the prior art. These and other advantages of the present invention will become apparent from the description that follows. [Means for solving the problem]

[0005] One aspect of the present invention provides a method for separating a virus from a fluid, the method comprising the steps of: (a) passing a fluid containing the virus through a porous filter comprising a porous medium at a differential pressure of at least 20 psid to obtain the virus; (b) reducing the pressure to 3 psid or less; and (c) passing an additional fluid containing the virus through the porous medium at a pressure of at least 20 psid to obtain the additional virus. DETAILED DESCRIPTION OF THE INVENTION

[0006] According to one embodiment of the present invention, there is provided a method for separating a virus from a fluid, the method comprising the steps of: (a) passing a fluid containing the virus through a porous filter comprising a porous medium at a differential pressure of at least 20 psid to obtain the virus; (b) reducing the pressure to 3 psid or less; and (c) passing an additional fluid containing the virus through a porous filter comprising a porous medium at a pressure of at least 20 psid to obtain the additional virus.

[0007] In one embodiment, the pressure in step (b) is 0 psid.

[0008] In typical embodiments, the reduced pressure in step (b) is for at least 2 minutes or at least 5 minutes, and / or the pressure in steps (a) and (c) is 30 psid or in the range of 20 psid to 40 psid. In some embodiments, the reduced pressure in step (b) is in the range of 2 minutes to 15 minutes.

[0009] In a preferred embodiment of the method, the isolated virus is an adeno-associated virus (AAV), which includes 11 AAV serotypes.

[0010] Advantageously, smaller viruses (e.g., AAV) can be separated (e.g., purified) from the fluid being filtered with minimal or no impact on the removal of larger adventitious viruses (e.g., viruses larger than about 50 nm, such as retroviruses). For example, the recovery of separated AAV (separated from larger viruses) can be increased by at least 1 log, and in some embodiments, by more than 2 logs, while mitigating the risk of contamination with larger viruses. With respect to the removal of large viruses, using PR772 (approximately 82 nm in size) as a representative large virus, the porous media typically achieves a large virus log reduction value (LRV) of at least 6 logs, calculated, for example, from the titer of phage content in the starting material and the pooled effluent.

[0011] Various porous filters comprising porous media, and filter devices including porous filters comprising porous media (including those commercially available; suitable filters may include two or more membranes / layers of porous media) can be used in accordance with embodiments of the present invention. The porous media can have any suitable pore structure, e.g., pore size (e.g., bubble point, or K, as described, for example, in U.S. Pat. No. 4,340,479). L or evidenced by capillary condensation flow porometry), pore rating, pore diameter (e.g., when characterized using a modified OSUF2 test as described in U.S. Pat. No. 4,925,572), or removal rate that allows the passage of one or more classes / types of smaller viruses of interest while minimizing / preventing the passage of undesirable materials and / or larger viruses of undesirable classes / types as fluid passes through the porous medium.

[0012] In a preferred embodiment, where the resulting smaller viruses are separated from larger viruses in the fluid, the size of the smaller viruses is less than 40 nanometers.

[0013] In a preferred embodiment, the porous medium (membrane) prevents the passage of viruses having a size of about 45 nanometers or greater.

[0014] Preferred filter devices, including porous filters comprising porous media suitable for use in embodiments of the present invention, include Ultipor® DV50VF grade virus removal filters (Pall Corporation, Port Washington, NY). Other suitable filter devices include those described, for example, in U.S. Patent Nos. 7,318,800 and 6,113,784.

[0015] The porous media may have, for example, any desired critical wetting surface tension (CWST, as defined, for example, in U.S. Pat. No. 4,925,572), and are additionally disclosed, for example, in U.S. Pat. Nos. 5,152,905, 5,443,743, 5,472,621, and 6,074,869. Typically, the media is water-wettable, e.g., having a CWST of 72 dynes / com (72×10 -5 N / cm) or more.

[0016] A variety of membranes are suitable for use as porous media in embodiments of the present invention, including commercially available membranes, such as those available from Pall Corporation (Port Washington, NY).

[0017] According to embodiments of the present invention, porous filters / porous media may have a variety of configurations, including planar (flat), pleated, hollow cylindrical, and hollow pleated cylindrical.

[0018] In some embodiments, a filter device can include, in addition to a porous filter, additional elements, layers, or components that may have different structures and / or functions, such as at least one of any one or more of pre-filtration, support, drainage, spacing, and buffering. Illustratively, a filter device, or a filter that includes a porous medium, can also include at least one additional element, such as a mesh and / or a screen.

[0019] The porous filter / porous medium is typically disposed within a housing having at least one inlet and at least one outlet, defining at least one fluid flow path between the inlet and outlet, with the porous medium or filter comprising the porous medium traversing the fluid flow path to provide a filter device. Preferably, the filter device is sterilizable. Any housing having a suitable shape and providing at least one inlet and at least one outlet can be employed.

[0020] The housing can be made of any suitable rigid, impermeable material that is compatible with the fluid being treated, including any impermeable thermoplastic material. Typically, the housing is made of a polymer. In some embodiments, the housing is a polymer, and in some embodiments, a transparent or translucent polymer such as acrylic, polypropylene, polystyrene, or polycarbonate resin. Such a housing is easy and economical to manufacture and allows for observation of the passage of fluid through the housing.

[0021] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0022] This example demonstrates that, according to one embodiment of the present invention, bacteriophage is utilized as a surrogate for AAV, increasing the isolation (and recovery) of smaller viruses while minimizing the presence of larger viruses in the filtered fluid.

[0023] An Ultipor® DV50 filter device (Pall Corporation, Port Washington, NY) containing porous media is obtained.

[0024] 10 in phosphate-buffered saline (PBS) containing 0.02% v / v Tween 80 7PFU / mL PR772 (size approximately 82 nm) and 10 7 Inoculate both PFU / mL PP7 (approximately 25 nm in size).

[0025] The inoculated fluid is mixed and a sample is taken to determine the pre-filtration level. The liquid is divided into two separate volumes so that it can be tested at two different test pressures (20 psi and 30 psi). The tests are performed as shown in Table 1 below:

[0026] [Table 1]

[0027] The post-filtration input sample is also removed to account for any loss of bacteriophage titer during the test. All aliquots are assayed for PR772 and PP7.

[0028] Larger bacteriophages (PR772, approximately 82 nm) are 100% retained at initial pressurization, after depressurization, and after subsequent pressurization.

[0029] After depressurization and subsequent repressurization, the yield of PP7 was observed to increase by approximately 1.7 logs at 20 psid and 30 psid.

[0030] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0031] The use of the terms "a" and "an," as well as "the," "at least one," and similar referents in the context of describing the invention (particularly in the context of the claims below) are to be construed as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") is to be construed as meaning one item selected from the listed items (A or B), or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. 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. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of individually referencing each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better clarify the invention and do not limit the scope of the invention unless otherwise asserted. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0032] Preferred embodiments of the present invention, including the best mode known to the inventors for carrying out the invention, are described herein. Variations of these preferred embodiments will be apparent to those of skill in the art upon reading the foregoing description. The inventors expect that skilled artisans will adopt such variations as appropriate, and the inventors intend to practice the invention otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. 1. A method for isolating viruses from a fluid, comprising: (a) passing a virus-containing fluid through a porous filter comprising a porous medium at a differential pressure of at least 20 psid to obtain the virus; (b) reducing the pressure to 3 psid or less; (c) passing the additional virus-containing fluid through the porous filter containing the media at a pressure of at least 20 psid to obtain additional virus; A method comprising:

2. 10. The method of claim 1, comprising separating larger viruses from smaller viruses from the fluid, wherein the resulting additional viruses are smaller viruses.

3. 3. The method according to claim 1, wherein the pressure in step (b) is 0 psid.

4. 4. The method according to claim 1, wherein in step (b), the reduced pressure is applied for at least 2 minutes.

5. 4. The method according to claim 1, wherein in step (b), the reduced pressure is applied for at least 5 minutes.

6. The method according to any one of claims 1 to 3, wherein in step (b), the reduced pressure is applied for a period ranging from 2 minutes to 15 minutes.

7. 7. The method of claim 1, wherein the pressure in steps (a) and (c) is at least 30 psid.

8. 7. The method according to claim 1, wherein the pressure in steps (a) and (c) ranges from 20 psid to 40 psid.

9. 9. The method of any one of claims 1 to 8, wherein the additional virus obtained is AAV.

Images