Effective removal of impurities using diafiltration

Abstract

Provided is a method for purifying the viral vector from a solution including a viral vector and a host cell protein (HCP). The method includes using a tangential flow filtration (TFF) mode with a load of a bioreactor harvest between an ultrafiltration / diafiltration membrane surface area per square meter to 100 liters, and under a pulsating flow with a frequency of 1.66 to 50 Hz and an amplitude of 2% to 25%, a diafiltration buffer is continuously added to circulate the solution through the ultrafiltration / diafiltration membrane. The method further includes filtering the solution through the ultrafiltration / diafiltration membrane to provide a permeate and a retentate and collecting the retentate so as to obtain a purified viral vector solution. The volume of the retentate is kept constant by continuously adding a diafiltration buffer. The viral vector is retained in the retentate. The HCP is filtered out by the permeate and the reduction of the HCP in the solution is between 1.5 and 4.3log.

Description

Technical Field

[0001] The present disclosure relates to the fields of biotechnology and medicine and, more particularly, to the purification of biological products by filtration. Background Art

[0002] Biological products such as proteins and viral vectors ideally contain low levels of chemical impurities. Viral vectors must be purified by removing host cell protein (HCP) impurities remaining in cell culture. In the field of recombinant viral vectors, for example, large-scale production and purification of pharmaceutical-grade viruses are required. Recombinant adenoviruses are a well-known class of viral vectors used for gene therapy and vaccination purposes.

[0003] After the virus is propagated in the cell, it is usually necessary to purify the virus for use in patients or vaccines. Purification methods in the prior art include, for example, chromatography and filtration. For example, in some purification methods, ultrafiltration / diafiltration steps can be used to concentrate the virus and / or exchange the buffer in which the virus is stored.

[0004] However, despite these prior art methods, there remains a need to develop efficient methods for viral vector purification that provide enhanced removal of impurities. Summary of the invention

[0005] Provided is a method for purifying a viral vector from a solution containing a viral vector and impurities (such as HCP). The method includes a) using a tangential flow filtration (TFF) mode with a load of a bioreactor harvest between 5 and 100 liters per square meter of ultrafiltration / diafiltration membrane surface area, and under a pulsating flow with a frequency of 1.66 to 50 Hz and an amplitude of 2% to 25%, continuously adding a diafiltration buffer to circulate the solution through the ultrafiltration / diafiltration membrane; b) filtering the solution through an ultrafiltration / diafiltration membrane to provide a permeate and a retentate; and c) collecting the retentate to obtain a purified viral vector solution. The volume of the retentate is kept constant by continuously adding a diafiltration buffer. The viral vector is retained in the retentate. The HCP is filtered out by the permeate and the reduction of the HCP in the solution is between 1.5log and 4.3log. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 is a schematic diagram of an ultrafiltration / diafiltration method according to an embodiment of the present invention;

[0007] Figure 2 depicts a cross-flow oscillating flow pattern of a portion of an ultrafiltration / diafiltration method according to an embodiment of the present invention; and

[0008] Figure 3 Depicted is a cross-flow stable flow pattern of a portion of an ultrafiltration / diafiltration process. DETAILED DESCRIPTION

[0009] A method for purifying a viral vector from a solution comprising the viral vector and impurities is provided.

[0010] Although the following discussion focuses on the use of the present invention in the purification of viral vectors, it will be appreciated that the method can be applied to a variety of biological materials.

[0011] Viruses can be propagated in cells (sometimes referred to as "host cells"). Cells are cultured to increase cell and virus numbers and / or viral titers. Cells are cultured to enable them to metabolize and produce the target virus. This can be achieved by methods well known to those skilled in the art.

[0012] Examples of viral vectors suitable for use with the present invention include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, poxvirus vectors, modified vaccinia Ankara (MVA) vectors, enterovirus vectors, Venezuelan equine encephalitis virus vectors, Semliki Forest virus vectors, tobacco mosaic virus vectors, lentiviral vectors, and the like.

[0013] In certain embodiments of the present invention, the vector is an adenovirus vector. The adenovirus according to the present invention belongs to the family Adenoviridae, and preferably belongs to the genus Mastadenovirus. It can be a human adenovirus, but it can also be an adenovirus that infects other species, including but not limited to bovine adenovirus (e.g., bovine adenovirus 3, BAdV3), canine adenovirus (e.g., CAdV2), porcine adenovirus (e.g., PAdV3 or PAdV5), or simian adenovirus (which includes monkey adenovirus and simian adenovirus, such as chimpanzee adenovirus or gorilla adenovirus). Preferably, the adenovirus is a human adenovirus (HAdV or AdHu) or a simian adenovirus such as a chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV). In the present invention, human adenovirus means if it is called Ad without specifying the species, for example, the abbreviated symbol "Ad26" means the same as HadV26, which is human adenovirus serotype 26. Also as used herein, the notation "rAd" means recombinant adenovirus, for example, "rAd26" means recombinant human adenovirus 26.

[0014] In certain preferred embodiments, the recombinant adenovirus according to the present invention is based on a human adenovirus. In a preferred embodiment, the recombinant adenovirus is based on human adenovirus serotypes 5, 11, 26, 34, 35, 48, 49, 50, 52, etc. According to a particularly preferred embodiment of the present invention, the adenovirus is human adenovirus serotype 26.

[0015] One of ordinary skill in the art will recognize that elements derived from multiple serotypes can be combined in a single recombinant adenoviral vector. Thus, chimeric adenoviruses can be generated that combine desirable properties from different serotypes. Thus, in some embodiments, the chimeric adenoviruses of the invention can combine the lack of pre-existing immunity of a first serotype with features such as temperature stability, assembly, anchoring, yield, redirected or improved infection, stability of DNA in target cells, and the like.

[0016] In certain embodiments, the recombinant adenoviral vector useful in the present invention is primarily or entirely derived from Ad26 (i.e., the vector is rAd26). The preparation of recombinant adenoviral vectors is well known in the art. The preparation of rAd26 vectors is described, for example, in WO 2007 / 104792 and in Abbink et al., (2007) Virol [Virology] 81(9):4654-63. An exemplary genomic sequence of Ad26 is found in GenBank Accession No. EF 153474 and in SEQ ID NO: 1 of WO 2007 / 104792. Examples of vectors for use in the present invention include, for example, those described in WO 2012 / 082918, the disclosure of which is incorporated herein by reference in its entirety.

[0017] However, it should be understood that the methods of the present invention are not limited to adenoviruses, but can be applied to a wide range of other viruses (e.g., adeno-associated viruses, poxviruses, iridoviruses, herpesviruses, papovaviruses, paramyxoviruses, orthomyxoviruses, retroviruses, vaccinia viruses, rotaviruses, flaviviruses) and other biological materials, such as proteins.

[0018] Biological products typically include various contaminants or impurities remaining in cell cultures. "Contaminants" or "impurities" are any ingredients in new drug products other than excipients in drug substances or drug products. The goal of the method of the present invention is to remove host cell proteins (HCPs), but other impurities may or may not be removed together with the removal of HCPs. Examples of such impurities include, but are not limited to, host cell DNA (HC-DNA), Triton X-100, Tris, sodium phosphate (monobasic and dibasic), magnesium chloride (MgCl2), HEPES, and insulin.

[0019] More specifically, after chemical lysis of the cell membrane, the virus (product) is released into the culture medium, and then the impurities in the lysed harvest material are flocculated. After removing these impurities, the material is clarified to be loaded onto the chromatographic membrane. The resulting material (viral vector) is a concentrated product, which also contains other impurities, such as HCP or HC-DNA.

[0020] According to the present invention, the viral vector is then subjected to ultrafiltration / diafiltration to remove impurities (such as HCPs remaining in the cell culture) to purify the viral vector. The preferred ultrafiltration / diafiltration method is tangential flow filtration.

[0021] refer to Figure 1 , a schematic diagram of an ultrafiltration / diafiltration method according to an embodiment of the present invention is provided. A feed tank 10 includes a sample solution to be filtered, such as a solution containing a target viral vector. The solution enters a filtration unit 12 through a feed channel or a feed line 14. Preferably, a first mechanical pump 16 is provided in the feed line 14 for circulating and controlling the flow of the solution. The filtration unit 12 includes an ultrafiltration / diafiltration membrane 18. As the feed solution is supplied to the filtration unit 12, the ultrafiltration / diafiltration membrane 18 separates the solution into a permeate and a retentate.

[0022] The diafiltration buffer is continuously added to the feed solution in the feed tank 10 by the diafiltration line 26 to maintain the total product (retentate) volume. A second pump 28 can be provided in the diafiltration buffer line 26 to control the supply of the diafiltration buffer to the feed tank 10. Any known buffer that does not affect the virus to be purified can be used. Preferably, the buffer has a pH of about 6.2 and contains small molecules to stabilize the product / virion. Preferably, 7 to 11 diafiltration volumes (DFV) are exchanged during the diafiltration step. More preferably, 10 DFV are exchanged during the diafiltration step.

[0023] In one embodiment, one or more detectors (not shown) may be provided in the feed line 14 to measure the pressure across the ultrafiltration / diafiltration membrane 18 .

[0024] The pressure difference across the ultrafiltration / diafiltration membrane 18 causes the feed solution, more particularly the impurities, to flow through the ultrafiltration / diafiltration membrane 18, so that the impurities are contained in the permeate. More particularly, the feed solution containing the viral vector passes through the ultrafiltration / diafiltration membrane 18, so that the impurities are removed from the feed solution and retained in the permeate, while the viral vector cannot pass through the ultrafiltration / diafiltration membrane 18 and is therefore retained in the retentate.

[0025] The surface area of ​​the ultrafiltration / diafiltration membrane 18 can be selected according to the volume of the feed solution to be purified. The ultrafiltration / diafiltration membrane 18 can have different pore sizes, depending on the purified biological material (e.g., viral vector) and the impurities contained therein. Preferably, the pore size of the ultrafiltration / diafiltration membrane 18 is small enough to retain the viral vector in the retentate, but large enough to effectively remove impurities in the permeate (i.e., allow impurities to pass through the membrane pores). For adenoviral vectors, the ultrafiltration / diafiltration method uses a nominal molecular weight limit (Nominal Molecular Weight Limit) (NMWL) in the range of 100 to 1,000 kilodaltons (kDa), preferably in the range of 300 to 500 kDa, more preferably 300 kDa membrane 18. Therefore, impurities, such as HCP (molecular mass of about 10 kDa to 200 kDa), can pass through the ultrafiltration / diafiltration membrane 18 (and be contained in the permeate), while virus particles larger than the pores are retained in the retentate by the ultrafiltration / diafiltration membrane 18. That is, the retentate contains the final product (virus).

[0026] The ultrafiltration / diafiltration membrane 18 may be made of, for example, regenerated cellulose, polyethersulfone, polysulfone, or derivatives thereof. The ultrafiltration / diafiltration membrane 18 may be of any known type or configuration, such as a flat plate or sheet, a spiral wound member, a tubular member, or a hollow fiber. In one embodiment of the present invention, the ultrafiltration / diafiltration membrane 18 is manufactured by MilliporeSigma. 2 ultrafiltration boxes.

[0027] The permeate leaves the filtration unit 12 through a permeate passage or permeate line 20 and is sent to a permeate collection tank 25. In one embodiment, as Figure 1 As shown, a third mechanical pump 22 is provided in the permeate line 20 to control the flow of the permeate through the permeate line 20. That is, the first pump 16 and the third pump 22 assist in the separation of impurities from the viral vector, the first pump 16 feeds and recirculates the feed solution and the retentate, and the third pump 22 promotes the removal of impurities (e.g., HCP) through the membrane pores and the permeate.

[0028] The retentate containing the viral vector of interest enters the retentate channel or retentate line 24, which is recycled back to the feed tank 10. The first pump 16 pumps at about 250 liters / m 2 The feed solution / retentate is supplied and recycled to and through the ultrafiltration / diafiltration membrane 18 at a flux of about 400 LMH / hour (LMH), and more preferably about 360 LMH. Preferably, the feed solution / retentate is maintained at a constant flow rate and volume. Preferably, the feed solution / retentate is maintained at a constant flow rate and volume per m 2 The bioreactor harvest with a membrane area between 5 L and 100 L, more preferably per m 2The loading of the bioreactor harvest is between 5 L and 60 L of membrane area, and most preferably per m 2 The loading of bioreactor harvest with membrane area between 5 L and 45 L supplies the feed solution / retentate to the ultrafiltration / diafiltration membrane 18 .

[0029] The operation of the third pump 22 and the flow rate of the permeate are a function of (i.e., are dependent upon) the operation of the first pump 16 and the flow rate of the feed solution / retentate. Preferably, the flow rate of the permeate is set to less than 20% of the feed solution / retentate, more preferably between 5% and 15%, and most preferably about 10%. Thus, when the feed solution / retentate is maintained at a flow rate between 250 and 400 LMH, the permeate is preferably maintained by the third pump 22 at a flow rate between 25 and 40 LMH, most preferably at a flow rate of 36 LMH (i.e., 10% of the target flow setpoint of 360 LMH for the feed solution / retentate).

[0030] In one embodiment, the first pump 16 is preferably a positive displacement pump. In one embodiment, the first pump 16 and the third pump 22 are both positive displacement pumps. Examples of positive displacement pumps that can be used include, for example, rotary cam pumps, screw pumps, rotary gear pumps, piston pumps, diaphragm pumps, screw pumps, gear pumps, hydraulic pumps, rotary vane pumps, peristaltic pumps, rope pumps, and flexible impeller pumps. In a preferred embodiment, the first pump 16 is a peristaltic pump. Preferably, the third pump 22 is also a peristaltic pump.

[0031] In a preferred embodiment, the ultrafiltration / diafiltration process is performed under an oscillating flow profile, wherein the oscillating flow profile results in a pulsating fluid action. Preferably, only the first pump 16 is operated under an oscillating flow profile, while the other pumps in the system are operated under a relatively stable (non-oscillating) flow profile, meaning that the flow profile may exhibit small amplitude oscillations but is relatively stable. However, other pumps, such as the third pump 22, may also be operated under an oscillating flow profile. For example, with Figure 3 Compared to the smoother, more stable flow pattern shown in FIG. 1 , the preferred oscillating flow pattern of this method is as shown in FIG. Figure 2 shown.

[0032] By flow oscillation (such as Figure 2 The pulsating fluid action caused by the ultrafiltration / diafiltration membrane 18 allows a larger amount of impurities (e.g., HCP) to pass through the ultrafiltration / diafiltration membrane 18 into the permeate (compared to the Figure 3 Preferably, greater than 1.5 log of impurities (e.g., HCP) are reduced by the ultrafiltration / diafiltration process of the present invention, and more preferably between 1.5 and 4.3 log of impurities are reduced, and most preferably between 1.5 and 2.3 log of impurities are reduced.

[0033] In a preferred embodiment, the first pump 16 is preferably operated to achieve a pulsating flow of a predetermined frequency and amplitude. More preferably, the pulsating flow of the first pump 16 has a frequency of 1.66 to 50 Hz, more preferably 1.66 to 33 Hz, and even more preferably 1.66 to 25 Hz. Preferably, the pulsating flow of the first pump 16 has a corresponding amplitude of 2% to 25%.

[0034] In particular, by conducting the ultrafiltration / diafiltration process under pulsating fluid action conditions with a frequency of 1.66 to 50 Hz and an amplitude of 2% to 25%, a reduction of impurities (e.g., HCP) greater than 1.5 log, and more particularly a reduction of 1.5 to 4.3 log, can be achieved. This is significantly greater than the reduction that can be achieved with conventional methods.

[0035] In one embodiment, the first pump 16 is also preferably operated to achieve a predetermined or target volumetric displacement. More preferably, the first pump 16 is operated to achieve a predetermined or target normalized displacement, expressed as volume per revolution per square meter of ultrafiltration / diafiltration membrane 18 surface area (ml / rev / m 2 ) indicates. In one embodiment according to the present invention, the first pump 16 is operated to achieve a flow rate of 10 to 100 mL / rev / m 2 range, and preferably between 17 and 83 mL / rev / m 2 range of normalized displacements, which results in superior impurity removal.

[0036] The ultrafiltration / diafiltration method according to an embodiment of the present invention is illustrated by the following, non-limiting example.

[0037] Invention Examples 1-10 : The adenovirus 26 viral vector anion exchange (AEX) chromatography eluate was processed. The eluate was divided into manageable batches and the eluate was separated into manageable batches under an oscillating flow pattern (i.e., pulsatile fluid action) and per m 2 Each batch of eluate was recirculated through a 300 kDa ultrafiltration / diafiltration membrane 18 at a constant flow rate of 360 LMH under a load of bioreactor harvest between 30 L and 40 L of membrane area. The permeate flow rate was maintained at 36 LMH. The frequency of the pulsating fluid action was in the range of 1.66 Hz to 50 Hz and its amplitude was in the range of 2% to 25%. In addition, the normalized displacement was maintained in the range of 17 to 83 ml per square meter of ultrafiltration / diafiltration membrane surface area per revolution. During the filtration of the eluate through the ultrafiltration / diafiltration membrane 18, the viral particles were retained in the retentate, while the HCP and other impurities were filtered out through the permeate. During the filtration method, buffer was added to the retentate to maintain the target total product volume. After exchanging 10 DFVs, the ultrafiltration / diafiltration method was completed. The method was performed multiple times using AEX eluates with different starting HCP concentrations.

[0038] Comparative Examples 1-12 : Under a stable flow pattern, the adenovirus 26 viral vector anion exchange (AEX) chromatography eluate is recirculated through a 300 kDa ultrafiltration / diafiltration membrane 18. During the filtration of the eluate through the ultrafiltration / diafiltration membrane 18, the viral particles are retained in the retentate, while HCP and other impurities are filtered out through the permeate. Buffer is added to the retentate to maintain the target total product volume. After exchanging 10 DFVs, the ultrafiltration / diafiltration method is completed. The method is performed multiple times using AEX eluates with different starting HCP concentrations, different recirculation flow rates, different permeate flow rates, and different retentate pressures.

[0039] Table 1 provides the results of these different experiments.

[0040] Table 1: HCP removal by ultrafiltration / diafiltration

[0041]

[0042] In the comparative examples summarized in Table 1, various process parameters, such as recirculation flow rate, permeate flow rate and retentate pressure, are varied while maintaining a stable flow pattern. Even with these other process parameters changes, effective removal of HCP (i.e., HCP reduction greater than 1.5 log and terminal HCP level less than 0.2 μg / mL) cannot be achieved. As shown in Table 1, only when the ultrafiltration / diafiltration process is carried out under an oscillating flow pattern can a reduction of HCP greater than 1.5 log (and more particularly a reduction of 1.5 to 4.3 log) be achieved. It is speculated that the pulsating fluid mechanism causes the gel layer of the lining ultrafiltration / diafiltration membrane 18 to be disturbed to a sufficient degree to allow HCP to pass through the membrane pores more easily than when the gel layer remains completely intact under the action of a non-pulsating fluid.

[0043] Further experiments were conducted under the conditions of Examples 1-3 of the present invention, some of which fell within the preferred ranges for the frequency and amplitude of the pulsating flow, while others fell outside the preferred ranges. These results are summarized in Table 2.

[0044] Table 2: Ultrafiltration / diafiltration method parameters

[0045]

[0046] *All RPM set points selected produce 360LMH retentate flow / flux.

[0047] From the results summarized in Table 2, it can be seen that when the frequency and amplitude of the pulsating flow are kept within the preferred range (i.e., a frequency of 1.66 to 50 Hz and an amplitude of 2% to 25%), the reduction in impurities is between 1.5 and 4.3 log. On the other hand, in the case where the frequency and / or amplitude falls outside the preferred range, such as in Run Nos. 5-9, the reduction in impurities is significantly lower.

[0048] According to the method of the present invention, no further purification is required after the ultrafiltration / diafiltration process. However, it should be understood that the product can optionally be further purified by methods known to those skilled in the art (such as density gradient centrifugation, chromatography, etc.).

[0049] Those skilled in the art will appreciate that changes may be made to the above embodiments without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A method for purifying a viral vector from a solution comprising a viral vector and a host cell protein (HCP), the method comprising: a) using tangential flow filtration (TFF) mode with a loading of between 5 and 100 liters of bioreactor harvest per square meter of ultrafiltration / diafiltration membrane surface area and circulating the solution through the ultrafiltration / diafiltration membrane under a pulsating flow having a frequency of 1.66 to 8.2 Hz and an amplitude of 2% to 25% with continuous addition of diafiltration buffer, and wherein the ultrafiltration / diafiltration membrane has a NMWL of 100 kDa to 500 kDa; b) filtering the solution through the ultrafiltration / diafiltration membrane to provide a permeate and a retentate, the volume of the retentate being kept constant by continuous addition of diafiltration buffer, the viral vector being retained in the retentate and the HCP being filtered out through the permeate, the reduction of the HCP in the solution being between 1.5 and 4.3 log; as well as c) collecting the retentate to obtain a purified viral vector solution, The ultrafiltration / diafiltration is carried out under an oscillating flow pattern.

2. The method of claim 1, wherein the viral vector is an adenoviral vector.

3. The method of claim 1, wherein the ultrafiltration / diafiltration membrane has a NMWL of 300 kDa.

4. The method of claim 1, wherein the flow rate of the solution to be filtered is 250 liters / m 2 / hour (LMH) to 400LMH.

5. The method of claim 4, wherein the flow rate of the solution to be filtered is 360LMH.

6. The method of claim 4, wherein the flow rate in LMH of the solution to be filtered is constant.

7. The method of claim 4, wherein the flow rate of the permeate is between 5% and 15% of the flow rate of the solution to be filtered.

8. The method of claim 1, wherein the filtering of the solution is performed using a peristaltic pump.

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