Patent Application
20200141912
The present invention relates to separation processes in the manufacturing of biopharmaceuticals, and more particularly to a separation process useful for separation of large species such as plasmids, virus particles etc.
In the manufacturing of biopharmaceuticals such as vaccines, antibodies, recombinant proteins, gene therapy vectors etc. several chromatographic separation steps are usually needed to remove various contaminants and impurities from the product. These separation steps add significant cost and process time and there is hence a significant interest in intensifying them.
Multicolumn continuous chromatography processes are available, where the feed is applied to a first column and is then diverted to one or more subsequent columns as the first columns approaches saturation and the first column is eluted and regenerated to be loaded again during elution and regeneration of the subsequent column(s). Such processes can be denoted periodic countercurrent chromatography (PCC) or simulated moving bed (SMB) and are of considerable interest for separation of biopharmaceuticals, see e.g. U.S. Pat. No. 7,901,581, US20130248451, US20130280788 and U.S. Pat. No. 7,220,356, which are hereby incorporated by reference in their entireties. PCC/SMB processes can significantly increase the productivity, but the increases achieved can depend quite strongly on the design of the process.
Plasmids are of interest in the biopharmaceutical area primarily as vectors for gene therapies and certain cell therapies (e.g. for preparing CAR-T cells) but also in vaccine technology. The plasmids are large ring-shaped DNA molecules of bacterial origin, which present particular issues in chromatographic processes as their size prevents them from entering the pores of most chromatography media. A common first step in plasmid purification is a so-called group separation, where the plasmid preparation is applied to a gel filtration resin and the plasmid is recovered in the void fraction or a slightly retained fraction, while RNA, a major contaminant, enters the pores of the resin and is significantly retained. As with other gel filtration processes, this is a slow process with limited loading capacity and it would be highly interesting to speed it up by applying continuous processing.
Accordingly, there is a need for new group separation processes for plasmids and other large species, such as e.g. virus particles, allowing an increased efficiency of the processes.
One aspect of the invention is to provide an efficient method for continuously separating a plasmid or e.g. a virus particle or other large species. This is achieved with a method of applying a process feed containing a plasmid/virus particle or other large species to an apparatus with at least three chromatography columns packed with separation matrix particles, wherein while one chromatography column is loaded with the process feed, another chromatography column is eluted with an eluent to recover the separated plasmid, and yet another chromatography column is eluted with a further eluent to remove contaminants.
Further suitable embodiments of the invention are described in the dependent claims.
As used herein, the terms “comprises,” “comprising,” “containing,” “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
In one aspect, the present invention discloses a method of continuous separation of a plasmid from a process feed comprising a plasmid in an apparatus with at least three chromatography columns. The method can also be used for other separations of large species, e.g. viruses, virus-like particles, antigen conjugates etc., from lower molecular-weight contaminants. The columns are packed with separation matrix particles, and while one chromatography column is loaded with the process feed, another chromatography column is eluted with an eluent to recover the separated plasmid, and yet another chromatography column is eluted with a further eluent (which can be the same eluent as used for eluting the plasmid) to remove contaminants such as RNA. It can be advantageous if the flow rates in the different steps are matched, so that no standstill is required in any step to wait for another step to finish.
The method may in some embodiments, as illustrated by
a) conveying a process feed comprising a plasmid through a first chromatography column comprising a packed bed of separation matrix particles;
b) conveying an eluent through the first chromatography column, recovering the eluent with the plasmid after passage of the first chromatography column; and conveying the process feed through a second chromatography column packed with the same separation matrix as the first chromatography column; and
c) conveying a further eluent (which can be the same eluent as in step b) through the first chromatography column; conveying an eluent through the second chromatography column, recovering the eluent with the plasmid after passage of the second chromatography column; and conveying the process feed through a third chromatography column packed with the same separation matrix as the first and second chromatography columns.
After step c), steps a)-c) may repeated for the duration of the continuous process, e.g. until the entire process feed has been processed. In these repetitions step a) may further comprise conveying a further eluent through the second chromatography column while conveying an eluent through the third chromatography column and recovering the eluent with the plasmid after passage of the third chromatography column; and step b) may further comprise conveying a further eluent through the third chromatography column. Suitably, a process feed or eluent is conveyed through all three columns throughout all of steps a)-c), i.e. no flows are stopped to wait for another column/step to finish.
In certain embodiments, as illustrated by
a) conveying a process feed comprising a plasmid through a first chromatography column comprising a packed bed of separation matrix particles, and conveying an outflow from the first chromatography column through a second chromatography column packed with the same separation matrix as the first column;
b) conveying an eluent through the first chromatography column, recovering the eluent with the plasmid after passage of the first chromatography column; and conveying the process feed through the second chromatography column and further through a third chromatography column packed with the same separation matrix as the first and second columns;
c) conveying a further eluent through the first chromatography column; conveying an eluent through the second chromatography column, recovering the eluent with the plasmid after passage of the second chromatography column; and conveying the process feed through the third chromatography column and further through a fourth chromatography column packed with the same separation matrix as the first, second and third columns; and
d) conveying a process feed comprising a plasmid through the fourth chromatography column and conveying an outflow from the fourth chromatography column through the first chromatography column; conveying a further eluent through the second chromatography column; and conveying an eluent through the third chromatography column, recovering the eluent with the plasmid after passage of the third chromatography column.
After step d), steps a)-d) may be repeated for the duration of the continuous process, e.g. until the entire process feed has been processed. In these repetitions, step a) may further comprise conveying a further eluent through the third chromatography column and conveying an eluent through the fourth chromatography column while recovering the eluent with the plasmid after passage of the fourth chromatography column; and step b) may further comprises conveying a further eluent through the fourth chromatography column. Suitably, a process feed or eluent is conveyed through all three columns throughout all of steps a)-d), i.e. no flows are stopped to wait for another column/step to finish.
The method can e.g. be carried out in a chromatography system 10 as illustrated in
In the embodiments discussed above, the separation matrix particles can suitably be gel filtration particles having an exclusion limit for dextrans of less than 5000 kDa, such as of 500 kDa-5000 kDa or 500-3000 kDa. These particles enable the separation of a plasmid, which can often be in the size range of 2-20 kb (2-7 kb for vaccine-type plasmids and up to 20 kb for plasmids to be used in gene therapy), from lower molecular weight contaminants such as the more or less degraded RNA present in cell lysates, typically having a size of <1 kb. The separation matrix particles may comprise a crosslinked polysaccharide, such as crosslinked agarose or alternatively crosslinked agar, cellulose or dextran. An example of crosslinked agarose particles can e.g. be Sepharose™ 6 Fast Flow beads (GE Healthcare Life Sciences) with a dextran exclusion limit of 2000 kDa, average particle diameter 90 μm and particle diameter range 45-165 μm. Alternatively, Sepharose CL-6B crosslinked agarose beads (GE Healthcare Life Sciences) with dextran exclusion limit approx. 1000 kDa may be used, although Sepharose 6 Fast Flow will allow higher flow rates due to its higher rigidity. To allow even higher flow rates, highly rigid crosslinked agarose beads may be used, e.g. beads as described in U.S. Pat. No. 6,602,990, hereby incorporated by reference in its entirety.
In some embodiments, the eluent (and optionally a further eluent) comprises at least 1.5 M of a salt, such as 1.5-2.5 M or 1.8-2.3 M salt. Alternatively expressed, the total salt concentration of the eluent (and optionally the further eluent) may be at least 1.5 M, such as 1.5-2.5 M or 1.8-2.3 M. The salt may e.g. be ammonium sulfate and the eluent/further eluent may e.g. comprise at least 1.5 M ammonium sulfate, such as 1.5-2.5 or 1.8-2.3 M ammonium sulfate. The presence of the salt/ammonium sulfate improves the separation between the plasmid and the RNA contaminants.
The process feed may comprise a clarified cell lysate, such as a lysate of gram negative cells, e.g. E. coli. In some embodiments, the method comprises, before the separation, a step 0) of preparing a clarified cell lysate as the process feed. Step 0) may comprise contacting cells, such as E. coli cells, with alkali and a surfactant, such as SDS, to induce lysis and neutralizing e.g. with potassium acetate. This is a well-known method, involving the addition of NaOH and SDS to final concentrations of e.g. 0.1 M and 0.05% (see e.g. L A Ciccolini et al: Biotech Bioeng 87, 293-302 (2004)). Step 0) may also be carried out in a continuous mode, e.g. by adding the lysis reagents in a static mixer or similar (see e.g. US2007/0213289, hereby incorporated by reference in its entirety). The clarification can be done by traditional filtration, but to avoid excessive clogging of filters, it can be advantageous to remove the bulk of the particulates by flocculation/flotation before a final filtration. This can be achieved by adding a hydrogen carbonate, e.g. ammonium hydrogen carbonate, to the crude neutralised lysate at a pH around 5, such as at pH 4-6 or 4.5-5.5. If the pH is outside this interval, it may be adjusted after the hydrogen carbonate addition so as to fall within the interval. The carbon dioxide bubbles generated under these conditions will cause flotation of flocculated particulates to the surface, leaving an almost totally clarified lysate as the bulk liquid. Such a flotation process can also be performed continuously, e.g. by flowing the lysate through a flotation vessel where the flotated material on the surface is scraped off or otherwise diverted, while the clarified lysate is passed through a filter without any clogging issues.
In addition to the steps discussed above, the method may further comprise, after the separation, a step e) of purifying the recovered plasmid. This can be done e.g. by bind-elute chromatography and may suitably involve separating the desired super-coiled (sc) plasmid from open circle (oc) and other conformations of the plasmid. Both thiophilic and hydrophobic interaction chromatography methods can be used for this purpose. For thiophilic chromatography, resins like PlasmidSelect™ or PlasmidSelect Xtra (GE Healthcare Life Sciences) may be used, while various chromatographic resins with e.g. phenyl, butyl or hexyl groups have been suggested for hydrophobic interaction chromatography of plasmids (see e.g. US2007/0213289, hereby incorporated by reference in its entirety). Step e) can also be performed in a continuous mode, e.g. by using PCC or SMB continuous chromatography techniques. This can be achieved by the direct application of eluate from the steps above to a second multicolumn chromatography system. Alternatively, in a system with several chromatography columns, some columns may be used for the initial separation and some columns may be used for the further purification. In this case, the system may comprise both columns packed with gel filtration resin and columns packed with e.g. thiophilic or hydrophobic interaction resin.
Sample: E. coli clarified lysate, containing plasmid pJV4 (6 kb), 17 mM Tris, 3.3 mM EDTA, 1 M potassium acetate.
Column: 20 ml Sepharose 6 Fast Flow (Sepharose 6FF) gel filtration resin (GE Healthcare Life Sciences) packed in a HiPrep™ 16/10 column (GE Healthcare Life Sciences), with 16 mm bed diameter and 100 mm bed height.
Chromatography system: ÄKTA pcc 75 system (GE Healthcare Life Sciences)
Equilibration buffer: 2.1 M ammonium sulfate, 10 mM EDTA, 100 mM Tris-HCl pH 7.5.
Elution buffer: 2.1 M ammonium sulfate, 10 mM EDTA, 100 mM Tris-HCl pH 7.5.
This was the initial run, for checking the separation pattern and starting optimisation. The results are shown in
The flow rates were increased in comparison with run 1. As shown in
A periodic countercurrent chromatography (PCC) setup with four columns of the same type as used in Example 1 was arranged in the ÄKTA pcc 75 chromatography system. The sample and the buffers were the same as in Example 1, although the equilibration step was only performed once for all the columns before starting the PCC cycles.
The results, as shown in
In this experiment, the amount loaded was slightly reduced to avoid elution in the flowthrough. Further, the step times were adjusted so that all steps could be run continuously without any waiting times. As above, the equilibration step was only run once, before the actual PCC cycles.
The results are shown in
This written description uses examples to disclose the invention, including the best mode, and also to enable any person skilled in the art to practice the invention, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal languages of the claims. Any patents or patent applications mentioned in the text are hereby incorporated by reference in their entireties, as if they were individually incorporated.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1710130.4 | Jun 2017 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/054478 | 2/23/2018 | WO | 00 |
