Method for selectively removing contaminants during the purification process of biological drugs - Patents.com

Abstract

The present invention relates to a method for selectively removing contaminants during the process of purifying a biological agent, comprising a step of filtering a solution containing (i) the biological agent and (ii) potential contaminants using a charge-neutral organic polymer filter in the presence of a kosmotropic agent, the one or more contaminants being contaminants that bind to the charge-neutral organic polymer filter in the presence of at least one kosmotropic agent, while the biological agent does not bind to the charge-neutral organic polymer filter in the presence of at least one kosmotropic agent. These methods combine in a single step the dual use of the filter for chemical and mechanical removal of contaminants. The present invention further relates to the respective methods for the purification of biological agents, as well as the respective uses of the charge-neutral organic polymer filter.

Description

[Technical field]

[0001] The present invention relates to a method for selectively removing contaminants during the process of purifying a biological agent, comprising a step of filtering a solution containing (i) the biological agent and (ii) potential contaminants using a charge-neutral organic polymer filter in the presence of a kosmotropic agent, the one or more contaminants being contaminants that bind to the charge-neutral organic polymer filter in the presence of at least one kosmotropic agent, while the biological agent does not bind to the charge-neutral organic polymer filter in the presence of at least one kosmotropic agent. These methods combine in a single step the dual use of the filter for chemical and mechanical removal of contaminants. The present invention further relates to the respective methods for the purification of biological agents, as well as the respective uses of the charge-neutral organic polymer filter. [Background technology]

[0002] Modern downstream bioprocessing is addressing the challenges in the purification of new classes of therapeutic modalities such as monoclonal antibodies (mAbs), plasmid DNA (pDNA), messenger RNA (mRNA), virus-like particles (VLPs) or nanoplexes as well as cell-based therapies (e.g. stem cell therapies). As a critical part of any current downstream processing scheme is based on chromatography, there is a constant need to improve purification efficiency and enhance the separation process.

[0003] Examples in this respect are the production of plasmid DNA used as gene therapy vectors or as a source of virus or mRNA production, which consists of multiple filtration and chromatography steps, as well as the production of recombinant proteins in, for example, E. coli.

[0004] Chromatography-based isolation of biological agents involves multiple process steps including, but not limited to, fermentation, lysis (in case of intracellular products or lysis of extracellular products can be avoided), clarification, capture chromatography and polishing chromatography (Figure 1). One example of such a process is the purification of plasmid DNA, which consists of the lysis of bacterial cells to release the target molecule (e.g., pDNA) as well as various impurities including host cell proteins, host cell DNA, host cell RNA and endotoxins that need to be removed by the purification process. Lysis is typically performed by the addition of strong base and detergent, followed by neutralization and precipitation of RNA using calcium chloride, and filtration using a non-adsorbent filter to remove particulates / precipitates (impurities) formed from the cell lysate upon precipitation of RNA.

[0005] Plasmids are then captured from the filtered and neutralized bacterial lysate on a weak anion exchange chromatography column, followed by polishing with hydrophobic interaction chromatography in the presence of a kosmotropic salt (e.g., (NH4)2SO4) to remove residual RNA, unwanted DNA isoforms, residual genomic DNA, residual endotoxins, and residual proteins (Figure 1).

[0006] The production of plasmid DNA is required as an independent gene therapy vector or vaccine, or as a source of material in the production of viruses, mRNA, circular RNA or other therapeutic modalities produced by in vitro transcription from a DNA template. There is a high demand for plasmid production and, as a result, pressure to increase the yield of the production / purification process.

[0007] Endotoxins are hydrophobic in nature, and host cell RNA and genomic (bacterial) DNA exhibit hydrophobic properties in the presence of kosmotropic agents commonly used in downstream purification, especially hydrophobic interaction chromatography. In addition to hydrophobic contaminants, the use of kosmotropic agents can lead to the precipitation of biomolecules, such as host cell proteins. The precipitates then lead to fouling of the chromatography column.

[0008] Contaminants such as host cell RNA, bacterial DNA and endotoxins usually bind to the surface of hydrophobic interaction chromatography resins and compete for binding with target molecules (e.g., pDNA), and selective elution from the HIC resin is achieved by adjusting the concentration of the kosmotropic agent. Thus, the presence of such contaminants reduces the binding capacity of the HIC column, potentially reducing the purity and / or yield of the target product.

[0009] Typically, pDNA elutes at higher salt concentrations than host cell RNA, but partial overlap in elution is common, resulting in reduced recovery of pDNA and it can be partially co-purified with RNA.

[0010] Host cell RNA and genomic DNA are typically removed by precipitation with CaCl2, but only partially. Residual host cell RNA, genomic DNA and endotoxins are removed by hydrophobic interaction chromatography and / or anion exchange chromatography using chromatographic devices such as porous particles, membranes or monoliths. Despite the application of multiple purification steps, contamination with residual host cell RNA, genomic DNA and endotoxins may result in insufficient purity to meet regulatory specifications.

[0011] The concentrations of proteins, RNA, bacterial DNA and endotoxins can be reduced by ammonium sulfate precipitation. This step also serves as a suitable conditioning step because the salt type and concentration (2M-2.5M) in the final pDNA-containing supernatant matches the optimal requirements for HIC feed. However, precipitation alone is not efficient enough to reduce contaminants below the specification limits. Summary of the Invention [Problem to be solved by the invention]

[0012] The technical problem underlying the present invention is therefore to provide means for improving the yield and / or purity in a purification process of a biological drug, in particular with regard to contaminating residual RNA, unwanted DNA isoforms, residues of genomic DNA, residual endotoxins and / or residual proteins. [Means for solving the problem]

[0013] The solution to the above technical problem is realized by the embodiments characterized in the claims.

[0014] In particular, in a first aspect, the present invention relates to a method for selectively removing one or more contaminants during a process for purifying a biological agent, comprising subjecting a solution containing said biological agent and potential contaminants to a filtration step using a neutral charge organic polymer filter in the presence of at least one kosmotropic agent, wherein said one or more contaminants are contaminants that bind to the neutral charge organic polymer filter in the presence of said at least one kosmotropic agent, whereas the biological agent does not bind to the neutral charge organic polymer filter in the presence of said at least one kosmotropic agent.

[0015] As used herein, the term "selective removal" refers to the fact that the removal of contaminants by the filtration step of the method of the present invention is selective for said contaminants, i.e., the filtration step removes said contaminants but does not remove any other components that may be present in the solution, or does not remove such other components to a substantial extent. In particular, the filtration step does not remove said biological agents, or does not remove said biological agents to a substantial extent. Preferably, at least 20% of the contaminants are removed, more preferably at least 50% of the contaminants, more preferably at least 80% of the contaminants, while preferably less than 5%, more preferably less than 2% of the other components are removed.

[0016] The term "contaminant" as used herein is not particularly limited and includes any contaminant that may be associated with a given process of purifying a biological agent, provided that the contaminant meets the above requirement of binding to the charge-neutral organic polymeric filter in the presence of the at least one kosmotropic agent, while the biological agent does not bind to the charge-neutral organic polymeric filter in the presence of the at least one kosmotropic agent. In other words, the contaminant exhibits a higher hydrophobicity in the presence of the at least one kosmotropic agent compared to the hydrophobicity of the biological agent in the presence of the at least one kosmotropic agent. In certain embodiments, the contaminant is a contaminant derived from the host cell, e.g., bacterial cell, in which the biological agent is produced. In further particular embodiments, the contaminant is selected from the group consisting of contaminating RNA species, host cell DNA (e.g., host cell genomic DNA, e.g., bacterial DNA), host cell proteins, endotoxins (i.e., bacterial lipopolysaccharide (LPS)), and mixtures thereof.

[0017] The term "during the process of purifying a biological agent" indicates that the method of the present invention can be carried out as part of a process of purifying a biological agent. The respective process is not particularly limited and includes any purification process in which contamination by contaminants may be relevant. By way of example, such processes include processes for purifying plasmid DNA (pDNA), processes for purifying recombinant proteins, and processes for purifying viral vectors, as known in the art.

[0018] The biological agent purified in the process of which the method of the present invention may be a part is not particularly limited and includes any biological agent in the purification process with which a contaminant may be associated. As noted above, in certain embodiments, the biological agent is selected from the group consisting of recombinant proteins, viral vectors, viral vaccines, pDNA, DNA-based therapeutics, and RNA-based therapeutics (e.g., mRNA). In preferred embodiments, the contaminant and the biological agent to be purified are not the same, i.e., the contaminant and the biological agent are not both proteins, not both DNA, or not both RNA.

[0019] In certain embodiments, the biological agent to be purified in a process of which the methods of the present invention may be a part is produced in bacterial cells (eg, Escherichia coli, etc.).

[0020] The charge-neutral organic polymer filter that can be used in the present invention is not particularly limited and includes any charge-neutral organic polymer filter known in the art, provided that the filter meets the requirement of binding impurities in the presence of the at least one cosmotropic agent, while not binding biological agents in the presence of the at least one cosmotropic agent. In other words, the charge-neutral organic polymer filter exhibits hydrophobicity in the presence of the at least one cosmotropic agent, specifically, exhibits hydrophobicity that allows binding of impurities to the filter but does not allow binding of biological agents to the filter. Preferably, such a charge-neutral organic polymer filter is selected from the group consisting of cellulose acetate filters, regenerated cellulose filters, polytetrafluoroethylene (PTFE) filters, polypropylene filters, polyethylene filters, polyamide filters, and unmodified polyethersulfone (PES) filters, with PES filters being particularly preferred. The pore size of each filter is not particularly limited and includes a pore size in the range of 0.2 μm to 50 μm, for example, 0.45 μm. By way of example, such membranes include Minisart 0.45 μm PES filters from Sartorius.

[0021] The present invention uses a neutrally charged organic polymeric filter in the presence of at least one kosmotropic agent, i.e. under specific hydrophobic interaction conditions (HIC), which allow selective binding of contaminants to the filter. Such HIC comprises the presence of a kosmotropic agent at a concentration of at least 0.5M, preferably at least 1M, more preferably 1M-3M, more preferably 2M-3M, for example about 2.5M. Furthermore, such HIC can comprise the further presence of NaCl at a concentration of at least 0.5M, preferably at least 1M, more preferably 1M-2M, for example about 1.5M. Furthermore, such HIC can further comprise a conductivity of at least 50 mSi / cm and / or a pH in the range of pH 6-pH 9, preferably pH 7-pH 8. Suitable kosmotropic agents are not particularly limited and are known in the art. Preferably, the cosmotropic agent is a cosmotropic salt of ammonium, potassium, cesium, sodium, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylate, and combinations thereof. In certain preferred embodiments, the cosmotropic agent is ammonium sulfate ((NH4)2SO4). In other certain preferred embodiments, the cosmotropic agent is sodium chloride (NaCl).

[0022] According to a preferred embodiment, the filtration step of the method of the present invention is performed before any HIC chromatography step that may be part of the process of purifying a biological agent. In this context, many conventional methods for the purification of biological agents include a step of hydrophobic interaction chromatography in the presence of kosmotropic salts using a chromatography column. Notably, these HIC chromatography steps are different from the filtration step of the method of the present invention, as the former uses a specific column material that does not have a filter functionality and does not use any charge-neutral organic polymer filter. Suitable HIC chromatography materials are not particularly limited and are known in the art. Preferably, the HIC chromatography material comprises a solid phase whose surface has butyl (C4), phenyl, octyl (C8), pyridine, or ether ligands.

[0023] In a preferred embodiment, the filtration step of the method of the present invention performs a dual function of mechanical filter and chemical adsorbent for contaminants that have a higher hydrophobicity than the target biological agent at a given concentration of at least one cosmotropic agent. Thus, the charge-neutral organic polymer filter simultaneously performs mechanical removal of particulates while functioning as a chemical adsorbent. This filtration step can be, but is not limited to, performed in-line with such HIC chromatography step, i.e., the filter outlet is connected to the inlet of the respective chromatography column. Preferably, the filtration step is performed immediately upstream of the HIC chromatography step, i.e., the filter outlet is directly connected to the inlet of the respective chromatography column.

[0024] In a second aspect, the present invention relates to a method for purifying a biological agent comprising carrying out a method according to the first aspect of the invention, thereby increasing the yield and / or purity of said biological agent.

[0025] In this embodiment, all relevant definitions and limitations given for the method according to the first aspect of the invention apply equally: in particular, contaminants, purification processes, biological agents, charge-neutral organic polymer filters, hydrophobic interaction conditions (HIC), and cosmotropic agents are as defined above for the first aspect of the invention.

[0026] In a third aspect, the present invention relates to the use of a neutrally charged organic polymer filter for the selective removal of contaminants during the process of purifying a biological agent, said neutrally charged organic polymer filter being used in the presence of at least one cosmotropic agent.

[0027] In this embodiment, all relevant definitions and limitations given for the method according to the first aspect of the invention apply equally: in particular, contaminants, purification processes, biological agents, charge-neutral organic polymer filters, hydrophobic interaction conditions (HIC), and cosmotropic agents are as defined above for the first aspect of the invention.

[0028] As used herein, the term "comprising" expressly includes the terms "consisting essentially of" and "consisting of," i.e., all of the above terms are interchangeable herein.

[0029] Furthermore, as used herein, the term "about" preferably refers to a modifier of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5% of the specified value. Thus, by way of example, the term "about 100" can include ranges of 90-110, 92-108, 94-106, 95-105, 96-104, 97-103, 98-102, 99-101, or 99.5-100.5.

[0030] The present invention provides a means for selectively removing contaminants during the process of purifying biological agents, whereby by means of cosmotropic agents used within a defined concentration window, host cell contaminants, such as RNA, endotoxins and residual genomic DNA, expel water from the surface and bind to the relatively hydrophobic surface of the charge-neutral organic polymer filter, while biological agents, such as pDNA monomers, do not exhibit sufficient hydrophobic behavior to bind to the filter and, as a result, pass through the filter. Thus, the contaminants are removed from the HIC load by the filter and the filtrate is applied directly to the hydrophobic interaction resin under hydrophobic interaction mode. This allows the biological agent to bind to the binding sites of the chromatography column, and if contaminants are present in the load, they will also bind to the column, thereby competing with the biological agent for the binding sites and reducing the binding capacity of the biological agent. Thus, filtration of the HIC load according to the present invention is an important contributor to high recovery of the target product, since it means that the contaminants do not bind to the surface of the chromatography resin, freeing up binding capacity for the binding of the biological agent.

[0031] The application of this unique feature of the charge-neutral organic polymer filter has multiple advantages for the economy and efficiency of downstream purification of biological agents. In particular, it provides a cost-effective purification approach for the removal of important contaminants that would otherwise be removed by chromatography, such as residual RNA, genomic DNA, and / or endotoxins. Furthermore, this prior removal of contaminants increases the recovery rate of HIC chromatography by widening the acceptable elution window of the biological agent. This may result in a wider range of acceptable conditions and a reduced number of rejected batches during production. Furthermore, this prior removal of contaminants increases the binding capacity of the HIC chromatography resin by increasing the binding surface available for biological agents that would otherwise be occupied by chemically related species. This reduces the size of the column required for the HIC chromatography step, i.e., the cost of the chromatography step. Furthermore, particulates formed in the presence of the cosmotropic agent are simultaneously mechanically removed by filtration, avoiding fouling of the HIC column.

[0032] Thus, the present invention advantageously increases the performance of downstream chromatography steps as follows: contaminants are removed by filtration in the presence of the kosmotropic agent, thereby increasing the recovery of the HIC chromatography step and, therefore, the overall process recovery. Additionally, aggregates in the feed stream upstream of the HIC chromatography step are removed by the filter, which functions in its traditional function as a mechanical filter.

[0033] In this context, neutrally charged organic polymer filters have traditionally been used in biological drug purification processes to remove particulates in a non-adsorptive manner (i.e., acting as a mechanical barrier for particles larger than the filter pores), with adsorption to the filter device typically being considered a drawback due to the possible removal of target molecules and the resulting reduction in process yield.

[0034] Thus, the present invention represents a counterintuitive use of charge-neutral organic polymer filters, such as PES filters. In particular, adsorption to such filters was considered a drawback by those skilled in the art due to the possibility of binding of target products. With the present invention, this drawback is turned into an advantage by combining several features that have not been applied so far to the purification of biological agents. In particular, the present invention takes a commonly used filter used for removing particulates, with the additional feature that the hydrophobic nature of the surface is enhanced by a matrix consisting of a high concentration of a kosmotropic agent.

[0035] A further unique feature of the present invention is the use of a filter immediately upstream of a commonly used hydrophobic interaction chromatography step. This is counterintuitive since filters are not typically used between two chromatography steps. In this case, the application of a filtration step in the presence of a cosmotropic agent is desirable for the purpose of chemical and simultaneous mechanical removal of contaminants.

[0036] Another unique feature of the present invention is that it does not require an additional buffer exchange step that would not be used in standard purification processes. The method requires the same buffer composition as the downstream hydrophobic interaction chromatography step. This is counterintuitive since PES filters are not expected to adsorb nucleic acid species under the experimental conditions used. [Brief description of the drawings]

[0037] [Figure 1] FIG. 1 shows a standard purification process scheme for the purification of biological agents, such as plasmid DNA. [Diagram 2] FIG. 1 shows the modification of a standard purification process scheme for the purification of biological agents, such as plasmid DNA, according to the present invention, namely, the inclusion of filtration under hydrophobic interaction conditions in the presence of a kosmotropic agent. [Diagram 3]Figure 1 shows an example of a HIC chromatography (polishing) step in the absence and presence of upstream PES filtration. A: C4 HLD (HIC) chromatography on plasmid DNA in the absence of PES filtration. B: C4 HLD (HIC) chromatography on plasmid DNA after PES filtration in the presence of the kosmotropic salt (NH4)2SO4. Arrows indicate the difference in elution profile in the absence / presence of filtration. FT: flow-through, E1-E3: elution fraction 1-elution fraction 3. [Figure 4] Agarose gel electrophoresis (AGE) of plasmid pAAV2 / 8 (7.3 kbp) fractions after the C4 HLD (HIC) chromatography step for case 1 and case 2. FT: flow-through, E1-E3: elution fractions E1-E3, 1: SC ladder (NEB), L-: load before PES filtration, L+: load after PES filtration, W: PES filter wash with ddHO, 2: Riboruler (ThermoFischer Scientific). [Diagram 5] Figure 2 shows HPLC chromatograms (CIMac pDNA) of plasmid pAAV2 / 8 (7.3 kbp) fractions after the C4 HLD chromatography step for Case 1 (A) and Case 2 (B). FT: flow-through, E1-E3: elution fractions E1-E3. [Figure 6] HPLC chromatogram (CIMac pDNA) of plasmid pAAV2 / 9n (7.3 kbp) fraction from C4 HLD chromatography step - sample preparation and filtration. PES filter wash: filtrate collected after washing the PES filter with ddH2O. Right: AGE of fractions 1: pre-PES load, 2: post-PES load, 3: PES filter wash. [Figure 7] HPLC chromatogram (CIMac pDNA) of plasmid pAAV2 / 5 (7.4 kbp) fraction from C4 HLD chromatography step - sample preparation and filtration. PES filter wash: filtrate collected after washing the PES filter with ddH2O. Right: AGE of fractions 1: pre-PES load, 2: PES filter wash, 3: post-PES load. [Figure 8] HPLC chromatogram (CIMac pDNA) of plasmid pUCBS4.7 (4.7 kbp) fraction from C4 HLD chromatography step - sample preparation and filtration. PES filter wash: filtrate collected after washing the PES filter with ddH2O. Right: AGE of fractions 1: pre-PES load, 2: post-PES load, 3: PES filter wash. [Figure 9] Figure 1 shows Limulus amebocyte lysate (LAL) assay results for endotoxin removal by PES filtration in the presence of kosmotropic agents. Plasmid samples were formulated under hydrophobic interaction (HIC) conditions (>1M AS or >1M NaCl). When filtered through a PES filter, LAL shows significant (>30-fold) removal of endotoxin. Endotoxin is released from the PES filter upon filter washing. Samples formulated under normal aqueous conditions show up to 2-fold endotoxin clearance (data not shown). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] The present invention is further illustrated by the following non-limiting examples. EXAMPLES

[0039] Materials and Methods: Plasmid DNA was prepared from E. coli cell paste up to the DEAE (diethylaminoethyl) elution step as known in the art. The DEAE eluate (50 mM Tris, 10 mM EDTA, approximately 0.75 M NaCl) containing plasmid DNA and contaminants was prepared for HIC chromatography with and without PES filtration as follows: Addition of MPC (see Table 1 below) to increase ammonium sulfate (AS) concentration to 2M Further dilution with MPL (2M AS) to further reduce the impact of NaCl from the DEAE eluate.

[0040] Case 1: C4 chromatography in the absence of PES filtration Load the prepared sample into the 2M AS via the system pump. Wash with MPL (2M AS) to return the UV signal to baseline (approximately 8 mL) A 3-minute linear gradient from MPL (2M AS) to MPA (no AS) 10CV ddH2O 10 CV 1M NaOH, Contact time in 1M NaOH: 15 min (see column preparation in Table 1 below)

[0041] Case 2: C4 chromatography in the presence of PES filtration The sample was prepared as in Case 1 with an additional filtration step before C4 loading as follows.

[0042] Prior to starting the run, samples formulated with 2M AS (see sample preparation above) were filtered through a Minisart™ PES syringe filter, 0.45 μm (Sartorius). The filter was then rinsed with MPL to improve pDNA recovery from the filter. After filtration, the filter was washed with ddH2O and the filtrate was collected as the PES filter wash.

[0043] For HPLC analysis, samples were diluted 4-fold with CIMac pDNA Analysis MPA (except for E3 and PES filter washes, which were diluted 2-fold) prior to injection to ensure sufficient reduction in AS concentration to ensure pDNA binding conditions.

[0044] [Table 1]

[0045] Results and Discussion: Comparison of the chromatograms of C4 chromatographic purification of Case 1 (Figure 3A) and Case 2 (Figure 3B) reveals differences in the UV signal for the flow-through (FT) between the unfiltered and PES-filtered C4 loads, indicating a higher impurity load in C4 in the absence of filtration.

[0046] The difference in the elution profile of the elution peaks can be explained as follows: in the absence of PES filtration, further impurities elute from C4 and are seen as an elution shoulder (E3 in Figure 3A). The peak is identified as RNA by agarose gel electrophoresis (see Figure 4 for case 1). As a result of the co-elution of impurities, the elution fraction of the plasmid (E2) should be collected separately from E3 to minimize contamination by RNA present in E3.

[0047] PES filtration prior to C4 chromatography eliminates the peak that elutes as E3 (Figure 3B), allowing us to expand the elution window of pDNA, thereby increasing the recovery of pure plasmid.

[0048] Agarose gel electrophoresis (AGE) (Figure 4) of fractions collected in case 1 and case 2 runs shows the prominent presence of RNA impurities eluting from the PES filter upon filter washing with ddHO (fraction W, Figure 4). Furthermore, the RNA signal is strong in E3 collected in case 1, whereas no RNA peaks are detected in the eluted fractions (E1-E3) of case 2.

[0049] In HPLC analysis of case 1 fractions (Figure 5A), RNA is detected in the CIMac pDNA column as a continuous, but not discontinuous, increase in signal between retention times 4 and 10 min (a heterogeneous population of short RNAs). The increase in RNA signal is only seen in the load, but not in the FT or wash, indicating that RNA binds to the C4 HLD. RNA elutes from C4 in E3. A very broad RNA peak co-elutes with the pDNA peak (oc, sc isoforms).

[0050] In case 2 (Figure 5B), the difference in RNA area between pre- and post-PES loading is not clearly discernible by HPLC. RNA binds to the PES filter in 2.0 M AS, and a large increase in RNA signal was observed in filter washes with ddH2O. A very broad RNA peak co-elutes with the pDNA peak (oc, sc isoforms). No RNA is seen in E3, and the elution of the sc plasmid isoform dominates.

Claims

1. 1. A method for selectively removing one or more contaminants during a process for purifying a biological agent, comprising: filtering the solution containing the biological agent and potential contaminants using a charge-neutral organic polymer filter in the presence of at least one kosmotropic agent; The method, wherein the one or more contaminants are contaminants that bind to the neutrally charged organic polymer filter in the presence of the at least one kosmotropic agent, while the biological agent does not bind to the neutrally charged organic polymer filter in the presence of the at least one kosmotropic agent.

2. 2. The method of claim 1, wherein the one or more contaminants are host cell-derived contaminants.

3. 3. The method of claim 1 or 2, wherein the one or more contaminants are selected from the group consisting of RNA, DNA, protein, endotoxin, and mixtures thereof.

4. 3. The method of claim 1 or 2, wherein the biological agent is selected from the group consisting of a recombinant protein, a viral vector, a viral vaccine, a plasmid DNA (pDNA), a DNA-based therapeutic, and an RNA-based therapeutic.

5. 3. The method of claim 1 or 2, wherein the charge-neutral organic polymer filter is selected from the group consisting of cellulose acetate filters, regenerated cellulose filters, polytetrafluoroethylene (PTFE) filters, polypropylene filters, polyethylene filters, polyamide filters, and unmodified polyethersulfone (PES) filters.

6. 3. The method of claim 1, wherein the at least one kosmotropic agent is present in the solution at a concentration of at least 0.5 M.

7. 7. The method of claim 6, wherein the solution exhibits a conductivity of at least 50 mSi / cm and / or a pH in the range of pH 6 to pH 9.

8. 3. The method of claim 1 or 2, wherein the cosmotropic agent is selected from the group consisting of kosmotropic salts of ammonium, potassium, cesium, sodium, fluoride, chloride, sulfate, carbonate, phosphate including pyrophosphate, carboxylate, and combinations thereof.

9. The kosmotropic salt is ammonium sulfate ((NH ４ ) ２ SO ４ 9. The method of claim 8, wherein the soluble salt is sodium chloride (NaCl).

10. 3. The method of claim 1 or 2, wherein the charge-neutral organic polymer filter simultaneously performs mechanical removal of particulates.

11. 3. The method of claim 1 or 2, wherein the filtration step occurs before any HIC chromatography step that may be part of the process for purifying the biological agent.

12. 12. The method of claim 11, wherein the filtration step is performed in-line with the HIC chromatography step.

13. 3. A method for purifying a biological agent, comprising carrying out the method according to claim 1 or 2, thereby increasing the yield and / or purity of said biological agent.

14. 1. Use of a charge-neutral organic polymer filter for the selective removal of contaminants in a process for purifying a biological agent, said filter being used in the presence of at least one kosmotropic agent.

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