Patent Application
20250179116
The present invention relates to methods for the selective removal of contaminants during a process of purifying a biologic agent, comprising a step of subjecting a solution containing (i) said biologic agent and (ii) potential contaminants to a filtration step using a charge-neutral organic polymer filter in the presence of a kosmotropic agent, wherein said one or more contaminants are contaminants that bind to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent, while the biologic agent does not bind to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent. These methods combine a dual use of the filter for chemical and mechanical removal of contaminants in a single step. The present invention further relates to respective methods for the purification of a biologic agent, as well as respective uses of a charge-neutral organic polymer filter.
Modern downstream bioprocessing addresses challenges in the purification of new classes of therapeutic modalities, such as monoclonal Antibodies (mAbs), plasmid DNA (pDNA), messenger RNA (mRNA), Virus Like Particles (VLP) or nanoplexes, as well as cell-based therapies (e.g. stem cell therapies). Since an important part of any current downstream processing scheme is based on chromatography, there is a constant need to improve the purification efficiency and to intensify the separation processes.
Examples in this respect are the production of plasmid DNA for use as a gene therapy vector, or as a raw material in production of viruses or mRNA, which consists of multiple filtration and chromatographic steps, as well as the production of recombinant proteins in e.g. E. coli.
The isolation of biologic agents based on chromatography comprises multiple process steps, including but not limited to fermentation, lysis (for intracellular product; or extracellular product lysis can be avoided), clarification, capture chromatography and polishing chromatography (
Plasmid is then captured from filtered neutralized bacterial lysate with a weak anion exchange chromatographic column, followed by polishing with hydrophobic interaction chromatography in the presence of kosmotropic salts (e.g. (NH4)2SO4) to remove residual RNA, unwanted DNA isoforms, remains of genomic DNA, residual endotoxin and residual proteins (
Production of plasmid DNA is required as a standalone gene therapy vector or a vaccine, or a raw material in 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 the production of plasmids, with a consequent pressure on increasing the yield of the production/purification process.
Endotoxins are inherently hydrophobic; host-cell RNA and genomic (bacterial) DNA exhibit hydrophobic properties in the presence of kosmotropic agents, which are commonly used in downstream purification, in particular hydrophobic interaction chromatography. In addition to hydrophobic contaminants, the use of kosmotropic agents can lead to precipitation of biologic molecules, e.g. host cell proteins. Precipitates in turn lead to fouling of chromatographic columns.
Contaminants such as host-cell RNA, bacterial DNA and endotoxins would normally bind to the surface of hydrophobic interaction chromatography resin and compete for binding with the target molecule (e.g. pDNA); selective elution from HIC resin is achieved by modulation of the concentration of kosmotropic agent. Presence of such contaminants therefore decreases the binding capacity of HIC columns, and potentially decreases purity and/or yield of the target product.
Typically, pDNA elutes at higher concentration of salt than host cell RNA, though partial overlap in elution is common, leading to lower recovery of pDNA, which can partly co-purify with RNA.
Host-cell RNA and genomic DNA are typically removed by precipitation by CaCl2, although the removal is partial. 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 multiple purification steps applied, contamination with residual host cell RNA, genomic DNA and endotoxins can lead to insufficient purity to meet regulatory specifications.
Proteins, RNA, bacterial DNA and endotoxin concentrations can be decreased with ammonium sulphate precipitation. This step further acts as a suitable conditioning step since the salt type and concentration (2-2.5 M) in the final pDNA-containing supernatant match the optimal requirements for a HIC feed. However, the precipitation alone is not sufficiently efficient to decrease the contaminants below the specification limits.
Accordingly, the technical problem underlying the present invention is the provision of means for improving the yield and/or purity in processes for the purification of biologic agents, in particular with respect to contaminating residual RNA, unwanted DNA isoforms, remains of genomic DNA, residual endotoxin and/or residual proteins.
The solution to the above technical problem is achieved by the embodiments characterized in the claims.
In particular, in a first aspect, the present invention relates to a method for the selective removal of one or more contaminants during a process of purifying a biologic agent, comprising a step of subjecting a solution containing said biologic agent and potential contaminants to a filtration step using a charge-neutral organic polymer filter in the presence of at least one kosmotropic agent, wherein said one or more contaminants are contaminants that bind to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent, while the biologic agent does not bind to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent.
As used herein, the term “selective removal” refers to the fact that removal of contaminants by way of the filtration step of the methods of the present invention is selective for said contaminants, i.e., said filtration step removes said contaminants but does not remove any other components that might be present in the above solution, or does not remove such other components to a substantial degree. In particular, the above filtration step does not remove the above biologic agent, or does not remove the biologic agent to a substantial degree. Preferably, at least 20% of contaminants, more preferably at least 50% of contaminants, more preferably at least 80% of contaminants are removed, while, preferably less than 5%, more preferably less than 2% of the above other components are removed.
The term “contaminants” as used herein is not particularly limited and includes any contaminants that might be relevant for a given process of purifying a biologic agent, provided said contaminants fulfil the above requirement of binding to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent, while the biologic agent does not bind to the charge-neutral organic polymer filter in the presence of said at least one kosmotropic agent. In other words, said contaminants exhibit a higher hydrophobicity in the presence of said at least one kosmotropic agent compared to the hydrophobicity of the biologic agent in the presence of said at least one kosmotropic agent. In specific embodiments, the contaminants are contaminants derived from the host cells in which the biologic agent is produced, e.g. from bacterial cells. In further specific embodiments, the contaminants are 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 lipopolysaccharides (LPS)), and mixtures thereof.
The term “during a process of purifying a biologic agent” indicates that the methods of the present invention can be performed as a part of a process of purifying a biologic agent. Respective processes are not particularly limited and include any purification processes in which contamination with contaminants may be relevant. By way of example, such processes include processes of purifying plasmid DNA (pDNA), processes of purifying recombinant proteins, and processes of purifying viral vectors, as known in the art.
The biologic agent to be purified in a process of which the methods of the present invention can be a part is not particularly limited and includes any biologic agents in the purification process of which contaminants may be relevant. As indicated above, in specific embodiments, the biologic 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 contaminants and the biologic agent to be purified are not the same, i.e., the contaminants and the biologic agent are not both proteins, are not both DNA, or are not both RNA.
In specific embodiments, the biologic agent to be purified in a process of which the methods of the present invention can be a part has been produced in bacterial cells (such as, e.g., in Escherichia coli).
Charge-neutral organic polymer filters that can be used in the present invention are not particularly limited and include any charge-neutral organic polymer filters known in the art, provided said filters fulfil the requirement of binding the contaminants in the presence of said at least one kosmotropic agent, while not binding the biologic agents in the presence of the at least one kosmotropic agent. In other words, the charge-neutral organic polymer filter exhibits hydrophobicity in the presence of said at least one kosmotropic agent, specifically a hydrophobicity that allows for the binding of the contaminants to the filter, but does not allow for the binding of the biologic agent to the filter. Preferably, such charge-neutral organic polymer filters are 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, wherein PES filters are particularly preferred. The pore size of respective filters is not particularly limited and includes pore sizes in the range of 0.2 μm to 50 μm, e.g., 0.45 μm. By way of example, such membranes include Sartorius Minisart 0.45 μm PES filters.
The present invention uses charge-neutral organic polymer filters in the presence of at least one kosmotropic agent, i.e., under specific hydrophobic interaction conditions (HIC), wherein these conditions enable the selective binding of the contaminants to the filter. Such HIC include the presence of a kosmotropic agent in a concentration of at least 0.5 M, preferably at least 1 M, more preferably of 1 to 3 M, more preferably 2 to 3 M, e.g. about 2.5 M. Further, such HIC can include the additional presence of NaCl in a concentration of at least 0.5 M, preferably at least 1 M, more preferably of 1 to 2 M, e.g. about 1.5 M. Moreover, such HIC can further include a conductivity of at least 50 mSi/cm and/or a pH in the range of pH 6 to pH 9, preferably pH 7 to pH 8. Suitable kosmotropic agents are not particularly limited and are known in the art. Preferably, the kosmotropic agent is a kosmotropic salt of ammonium, potassium, cesium, sodium, fluorides, chlorides, sulfates, carbonates, phosphates including pyrophosphates, carboxylates, and combinations thereof. In a specific preferred embodiment, the kosmotropic agent is ammonium sulfate ((NH4)2SO4). In another specific preferred embodiment, the kosmotropic agent is sodium chloride (NaCl).
According to preferred embodiments, the filtration step of the methods of the present invention is performed prior to any HIC chromatography steps that might be part of the process of purifying the biologic agent. In this context, many conventional methods for the purification of biologic agents contain a step of hydrophobic interaction chromatography in the presence of kosmotropic salts using chromatography columns. Of note, these HIC chromatography steps are different from the filtration step of the methods of the present invention, in that the former use specific column materials without filter functionality and not any charge-neutral organic polymer filters. Suitable HIC chromatography materials are not particularly limited and are known in the art. Preferably, the HIC chromatography material includes solid phases with surfaces possessing a butyl (C4), phenyl, octyl (C8), pyridine, or ether ligand.
In preferred embodiments, the filtration step of the methods of the present invention performs a dual function of mechanical filter and chemical adsorber of contaminants with a hydrophobicity greater than the target biologic agent at a given concentration of the at least one kosmotropic agent. Thus, the charge-neutral organic polymer filter simultaneously performs mechanical removal of particulates while acting as the chemical adsorber. This filtration step can be, but is not limited to being performed in-line with such HIC chromatography steps, i.e., the filter outlet is connected to the inlet of a respective chromatography column. Preferably, said filtration step is performed immediately upstream of said HIC chromatography steps, i.e., the outlet of the filter is directly connected to the inlet of a respective chromatography column.
In a second aspect, the present invention relates to a method for the purification of a biologic agent, comprising the step of performing the method according to the first aspect of the present invention, thereby increasing the yield and/or purity of said biologic agent.
In this aspect, all relevant definitions and limitations given for the methods according to the first aspect of the present invention equally apply. In particular, the contaminants, purification processes, biologic agents, charge-neutral organic polymer filters, hydrophobic interaction conditions (HIC), and kosmotropic agents are as defined above for the first aspect of the present invention.
In a third aspect, the present invention relates to the use of a charge-neutral organic polymer filter for the selective removal of contaminants during a process of purifying a biologic agent, wherein said charge-neutral organic polymer filter is used in the presence of at least one kosmotropic agent.
In this aspect, all relevant definitions and limitations given for the methods according to the first aspect of the present invention equally apply. In particular, the contaminants, purification processes, biologic agents, charge-neutral organic polymer filters, hydrophobic interaction conditions (HIC), and kosmotropic agents are as defined above for the first aspect of the present invention.
As used herein, the term “comprising”/“comprises” expressly includes the terms “consisting essentially of”/“consists essentially of” and “consisting of”/“consists of”, i.e., all of said terms are interchangeable with each other herein.
Further, as used herein, the term “about” preferably represents a modifier of the specified value of ±10%, more preferably ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, or ±0.5%. Thus, by way of example, the term “about 100” can include the ranges of 90 to 110, 92 to 108, 94 to 106, 95 to 105, 96 to 104, 97 to 103, 98 to 102, 99 to 101, or 99.5 to 100.5.
The present invention provides means for the selective removal of contaminants during a process of purifying a biologic agent, wherein kosmotropic agents used within a defined concentration window lead the host cell contaminants, e.g. RNA, endotoxins and residual genomic DNA to expel water from the surface and bind to the relatively hydrophobic surface of a charge-neutral organic polymer filter, while the biologic agent, e.g. pDNA monomers, do not exhibit sufficiently hydrophobic behavior to bind to the filter, consequently passing through the filter. Contaminants are thus removed from the HIC load by the filter; filtrate is applied directly onto a hydrophobic interaction resin, under hydrophobic interaction mode. This leads to binding of the biologic agent onto the binding sites of the chromatographic column; if contaminants were present in the load, these would also bind to the column, thereby competing with the biologic agent for binding sites and decreasing the binding capacity for the biologic agent. Filtration of HIC load according to the present invention is thus an important contributor to high recovery of target product, since it means that the contaminants do not bind to the surface of the chromatography resin and frees the binding capacity for binding of the biologic agent.
Applying this unusual feature of charge-neutral organic polymer filters has multiple benefits for the economics and efficiency of the downstream purification of biologics. In particular, it provides a cost-effective purification approach for removal of a critical contaminant that would otherwise be removed by chromatography, e.g. residual RNA, genomic DNA, and/or endotoxins. Further, due to prior removal of this contaminant, recovery of HIC chromatography is increased by extending the acceptable elution window of the biologic agent. This leads to broader acceptance conditions and potentially lower number of failed batches during production. Furthermore, due to prior removal of this contaminant, binding capacity of HIC chromatographic resin is increased by increasing the binding surface available for the biologic agent which would otherwise be occupied by chemically related species. This decreases the size of the column required for HIC chromatography step, i.e., decreases the cost of the chromatography step. Moreover, particulates formed in the presence of kosmotropic agents are concurrently mechanically removed by filtration, avoiding HIC column fouling.
Thus, the present invention advantageously increases the performance of the downstream chromatography step as follows: contaminants are removed by filtration in the presence of a kosmotropic agent, thereby increasing the recovery of HIC chromatography step, and consequently increasing the total process recovery. Additionally, aggregation of feed-stream upstream of the HIC chromatography step is removed by the filter functioning in its conventional function as a mechanical filter.
In this context, charge-neutral organic polymer filters are conventionally used in purification processes for biologic agents for the removal of particulates (i.e., acting as a mechanical barrier for particles larger than the filter pores) in a non-adsorbing fashion, wherein adsorption onto a filter device was typically considered a disadvantage, due to potential removal of target molecule and consequent lowering of process yield.
Thus, the present invention represents a counterintuitive use of charge-neutral organic polymer filters, such as e.g. PES filters. In particular, the adsorption onto such filters was considered a disadvantage by persons skilled in the art, due to potential binding of target product. By way of the present invention, this disadvantage is turned into an advantage by combining several features that have not been previously applied in the purification of biologic agents. Specifically, the present invention employs a commonly used filter used for the removal of particulates, with an added feature that the hydrophobic nature of the surface is augmented by matrices comprised of kosmotropic agents at high concentrations.
A further unusual feature of the invention is the use of the filter immediately upstream of a commonly used hydrophobic interaction chromatography step. This is counterintuitive because filters are not typically used between two chromatography steps. In this case, application of a filtration step in the presence of kosmotropic agents is desirable for purpose of chemical, as well as concurrent mechanical, removal of contaminants.
Another unusual feature of the present invention is that it requires no additional buffer exchange steps that would not be used in a standard purification process. The method requires buffer compositions identical to downstream hydrophobic interaction chromatography step. This is counterintuitive because PES filters are not expected to adsorb nucleic acid species under the experimental conditions used.
The figures show:
Standard purification process scheme for purification of biologics, e.g. plasmid DNA.
Modification of a standard purification process scheme for purification of biologics, e.g. plasmid DNA, by including the present invention, i.e., filtration under hydrophobic interaction conditions in the presence of kosmotropic agents.
Examples of HIC chromatography (polishing) step in the absence and presence of upstream PES filtration.
A: C4 HLD (HIC) chromatography on plasmid DNA in absence of PES filtration.
B: C4 HLD (HIC) chromatography on plasmid DNA after PES filtration in presence of kosmotropic salt (NH4)2SO4. Arrows indicate differences in elution profiles in absence/presence of filtration. FT: flow-through, E1-E3: elution fractions 1-3.
Agarose gel electrophoresis (AGE) of plasmid pAAV2/8 (7.3 kbp) fractions after Case 1 and Case 2 C4 HLD (HIC) chromatographic steps. 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 ddH2O, 2: Riboruler (ThermoFischer Scientific).
HPLC chromatograms (CIMac pDNA) of plasmid pAAV2/8 (7.3 kbp) fractions after Case 1 (A) and Case 2 (B) C4 HLD chromatographic steps. FT: flow-through, E1-E3: elution fractions E1-E3.
HPLC chromatograms (CIMac pDNA) of plasmid pAAV2/9n (7.3 kbp) fractions from C4 HLD chromatographic step—sample preparation and filtration. PES filter wash: filtrate collected after PES filter was washed with ddH2O. Right: AGE of fractions 1: load before PES, 2: load after PES, 3: PES filter wash.
HPLC chromatograms (CIMac pDNA) of plasmid pAAV2/5 (7.4 kbp) fractions from C4 HLD chromatographic step—sample preparation and filtration. PES filter wash: filtrate collected after PES filter was washed with ddH2O. Right: AGE of fractions 1: load before PES, 2: PES filter wash, 3: load after PES.
HPLC chromatograms (CIMac pDNA) of plasmid pUCBS4.7 (4.7 kbp) fractions from C4 HLD chromatographic step—sample preparation and filtration. PES filter wash: filtrate collected after PES filter was washed with ddH2O. Right: AGE of fractions 1: load before PES, 2: load after PES, 3: PES filter wash.
Limulus amoebocyte lysate (LAL) test results for removal of endotoxins by PES filtration in presence of kosmotropic agent. Plasmid sample was formulated in hydrophobic interaction (HIC) conditions (>1 M AS or >1 M NaCl). When filtered through PES filter, LAL shows significant (>30×) removal of endotoxins. Endotoxins are released from PES filter upon filter wash. Samples formulated under normal aqueous conditions show at best 2× endotoxin clearance (data not shown).
The present invention will be further illustrated by the following example without being limited thereto.
Plasmid DNA was prepared from E. coli cell paste up to DEAE (diethylaminoethyl) eluate stage as known in the art. DEAE eluate containing plasmid DNA and contaminants (in 50 mM TRIS, 10 mM EDTA, ca. 0.75 M NaCl) was prepared for HIC chromatography in the presence and absence of PES filtration as follows:
Sample was prepared as for Case 1, with an additional filtration step prior to C4 load as follows:
Before starting a run, the sample formulated in 2 M AS (see sample preparation above) was filtered through Minisart® PES Syringe Filter, 0.45 μm, Sartorius. Filter was then rinsed with MPL to improve pDNA recovery from the filter. After filtration, the filter was washed with ddH2O and filtrate collected as PES filter wash.
For HPLC analytics, samples were diluted 4× (with exception of E3 and PES filter wash, which were diluted 2×) with MPA for CIMac pDNA analytics before injection to reduce AS conc. enough to ensure binding conditions for pDNA.
Comparison of chromatograms of C4 chromatographic purification of Case 1 (
The difference in elution profile of elution peaks can be explained as follows: in the absence of PES filtration, additional impurity elutes from C4, seen as an elution shoulder (E3 in
PES filtration prior to C4 chromatography leads to an absence of a peak eluting as E3 (
Agarose gel electrophoresis (AGE) of fractions collected for runs Case 1 and Case 2 (
In HPLC analytics of Case 1 fractions (
In Case 2 (
| Number | Date | Country | Kind |
|---|---|---|---|
| 22158601.9 | Feb 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/053937 | 2/16/2023 | WO |
