INTENSIFIED VIRUS FILTRATION USING DIAFILTRATION BUFFER

Abstract

Method and system for purifying a sample comprising a biomolecule of interest and impurities, comprising expressing said biomolecule of interest in a bioreactor to form a product sample comprising said biomolecule of interest and impurities; subjecting said product sample to filtration to form a clarified product sample; subjecting said clarified product sample to affinity chromatography to remove impurities; subsequently subjecting said product sample to diafiltration followed by virus filtration and optional concentration. The buffer used during the diafiltration step (and thus in the virus filtration step) is the buffer desired for the final formulation of the product.

Claims

1. A method for purifying a sample comprising a biomolecule of interest and impurities, comprising expressing said biomolecule of interest in a bioreactor to form a product sample comprising said biomolecule of interest and impurities; subjecting said product sample to filtration to form a product sample; subjecting said product sample to affinity chromatography to remove impurities from said product sample, and subsequently subjecting said product sample to diafiltration prior to virus filtration.

2. The method of claim 1, wherein said affinity chromatography comprises Protein A affinity ligand.

3. The method of claim 1, further comprising subjecting said concentrated product sample to a virus inactivation step upstream of said diafiltration.

4. The method of claim 3, further comprising subjecting said c product sample to a polishing step downstream of said virus inactivation step and upstream of said diafiltration.

5. The method of claim 4, wherein said polishing step comprising one or more of anion exchange chromatography, cation exchange chromatography, and hydrophobic interaction chromatography.

6. The method of claim 1, wherein the biomolecule is an antibody selected from the group consisting of a recombinant antibody, a recombinant monoclonal antibody, a polyclonal antibody, a humanized antibody and an antibody fragment.

7. The method of claim 1, wherein said biomolecule is a protein.

8. The method of claim 1, wherein said biomolecule is a virus.

9. The method of claim 1, wherein said diafiltration step includes a diafiltration buffer, and wherein said virus filtration includes a virus filtration a virus filtration buffer having substantially the same composition as said diafiltration buffer.

10. A system for purifying a biomolecule of interest, comprising: a. a bioreactor; b. a filtration unit downstream of said bioreactor for clarifying the product sample exiting from said bioreactor; c. at least two affinity chromatography columns configured in series downstream of said filtration unit for receiving the clarified product stream from said filtration unit; d. a virus inactivation filter positioned downstream of said at least two affinity chromatography columns; e. one or more anion exchange, cation exchange or hydrophobic interaction exchange chromatography columns positioned downstream of said virus inactivation filter; f. a diafiltration unit positioned downstream of said one or more anion exchange, cation exchange or hydrophobic interaction exchange chromatography columns; and g. a virus filtration unit positioned downstream of said diafiltration unit.

11. The system of claim 10, wherein said at least two affinity chromatography columns each comprise Protein A affinity ligand.

12. The system of claim 10, configured to operate in a batch mode.

13. The system of claim 10, configured to operate in a continuous mode.

14. A system for purifying a biomolecule of interest, comprising: a. a bioreactor; b. a filtration unit downstream of said bioreactor for clarifying the product sample exiting from said bioreactor; c. at least two affinity chromatography membrane units configured in series downstream of said filtration unit for receiving the clarified product stream from said filtration unit; d. a virus inactivation filter positioned downstream of said at least two affinity chromatography membrane units; e. one or more anion exchange, cation exchange or hydrophobic interaction exchange chromatography membrane unit(s) positioned downstream of said virus inactivation filter; f. a diafiltration unit positioned downstream of said one or more anion exchange, cation exchange or hydrophobic interaction exchange chromatography membrane unit(s); and g. a virus filtration unit positioned downstream of said diafiltration unit.

15. The system of claim 14, wherein said at least two affinity chromatography units each comprise Protein A affinity ligand.

16. The system of claim 14, configured to operate in a batch mode.

17. The system of claim 14, configured to operate in a continuous mode.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a schematic representation of a conventional purification process used in the industry; and

[0022] FIG. 2 is a schematic representation of a purification system in accordance with certain embodiments.

DETAILED DESCRIPTION

[0023] In the following description, the terms “selected biomolecule”, “target biomolecule” or “molecule”, “target protein”, “biomolecule or protein of interest”, or similar terms all refer to products of a biomolecule manufacturing process.

[0024] The terms “contaminant,” “impurity,” and “debris,” as may be used interchangeably herein, refer to any foreign or objectionable molecule, including a biological macromolecule such as a DNA, an RNA, one or more host cell proteins, endotoxins, lipids, protein aggregates and one or more additives which may be present in a sample containing the product of interest that is being separated from one or more of the foreign or objectionable molecules. Additionally, such a contaminant may include any reagent which is used in a step which may occur prior to the separation process.

[0025] As used herein, the term “sample” refers to any composition or mixture that contains a target molecule, such as a target protein, to be purified. Samples may be derived from biological or other sources. Biological sources include eukaryotic and prokaryotic sources, such as plant and animal cells, tissues and organs. In some embodiments, a sample includes a biopharmaceutical preparation containing a protein of interest to be purified. In a particular embodiment, the sample is a cell culture feed containing a protein of interest to be purified. The sample may also include diluents, buffers, detergents, and contaminating species, debris and the like that are found mixed with the target protein or protein of interest. The sample may be “partially purified” (i.e., having been subjected to one or more purification steps, such as filtration steps) or may be obtained directly from a host cell or organism producing the target molecule (e.g., the sample may comprise harvested cell culture fluid).

[0026] As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed subject matter. The term permits the inclusion of elements or steps which do not materially affect the basic and novel characteristics of the apparatus, system or method under consideration. Accordingly, the expressions “consists essentially of” or “consisting essentially of” mean that the recited embodiment, feature, component, step, etc. must be present and that other embodiments, features, components, steps, etc., may be present provided the presence thereof does not materially affect the performance, character or effect of the recited embodiment, feature, component, step, etc. The presence of an operation or step that has no material effect on the sample or product is permitted. For example, a method consisting essentially of purifying a sample comprising a biomolecule of interest and impurities, consisting essentially of expressing said biomolecule of interest in a bioreactor to form a product sample comprising said biomolecule of interest and impurities; subjecting said product sample to filtration to form a clarified product sample; subjecting said concentrated product sample to affinity chromatography to remove impurities from said concentrated product sample; subjecting said product sample to virus inactivation; subjecting said product sample to purification/polishing such as with ion exchange chromatography; subjecting said product sample to buffer exchange by diafiltration, followed by subjecting said sample to virus filtration, excludes other steps or unit operations carried out between the bioreactor and the virus filtration operations, particularly between the diafiltration and virus filtration, that would materially change the composition of the product sample.

[0027] In certain embodiments, the sample that is the starting material of the process may vary depending upon the cell line in which it was grown as well as the conditions under which it is grown and harvested. For example, in most CHO cell processes the cells express the molecule outside of the cell wall into the media. One tries not to rupture the cells during harvest in order to reduce the amount impurities in the mixture. However, some cells during growth and harvesting may rupture due to shear or other handling conditions or die and lyse, spilling their contents into the mixture. In bacteria cell systems, the biomolecule is often kept with the cellular wall or it may actually be part of the cellular wall (Protein A). In these systems the cell walls need to be disrupted or lysed in order to recover the biomolecule of interest.

[0028] The target molecule to be purified can be any biomolecule, preferably a protein, in particular, recombinant protein produced in any host cell, including but not limited to, Chinese hamster ovary (CHO) cells, Per.C6® cell lines available from Crucell of the Netherlands, myeloma cells such as NSO cells, other animal cells such as mouse cells, insect cells, or microbial cells such as E. coli or yeast. Additionally, the mixture may be a fluid derived from an animal modified to produce a transgenic fluid such as milk or blood that contains the biomolecule of interest. Optimal target proteins are antibodies, immunoadhesins and other antibody-like molecules, such as fusion proteins including a C.sub.H2/C.sub.H3 region. For example, this product and process can be used for purification of recombinant humanized monoclonal antibodies such as (RhuMAb) from a conditioned harvested cell culture fluid (HCCF) grown in Chinese hamster ovary (CHO) cells expressing RhuMAb.

[0029] In certain embodiments, in the downstream purification process, a series of purification media having the desired chemical functionalities, are used to effect the removal of soluble impurities while the product remains in solution and flows through the purification media, resulting in a purified stream containing the product. Suitable forms of purification media include derivatized membranes, functionalized chromatography media, or any other porous material having the desired chemical functionality to interact with the various impurities so that the media can capture the impurities by electrostatic, hydrophobic, or affinity interactions. In view of the complex and varied natured of the impurities, a multitude of purification media having different chemical functionalities can be arranged in series to remove a variety of impurities having different chemical properties.

[0030] In certain embodiments, disclosed is a process for purifying a target molecule from a sample, where the process comprises: (a) expressing a protein in a bioreactor to form a protein sample; (b) subjecting the protein sample to filtration such as depth filtration; (c) subjecting the resulting protein sample to Protein A affinity chromatography, which employs one or more affinity chromatography units.

[0031] Also disclosed is a system for purifying a target molecule from a sample, comprising a bioreactor; a filter unit such as a depth filtration unit; one or more affinity chromatography columns such as one or more Protein A affinity chromatography columns in fluid communication with the filtration unit; optionally one or more virus inactivation units downstream of the one or more affinity chromatography columns; optionally a polishing phase downstream of the affinity chromatography columns, which may include one or more of an anion exchange chromatography column, a cation exchange chromatography column, and a hydrophobic interaction chromatography column; a diafiltration unit; a virus filtration unit; and an ultrafiltration unit downstream of the virus filtration unit.

[0032] In some embodiments, there is a connecting line between the various devices in the system. The devices are connected in line such that each device in the system is in fluid communication with devices that precede and follow the device in the system.

[0033] In some embodiments, the bioreactor used in a system according to the present invention is a disposable or a single use bioreactor. In some embodiments, the system is enclosed in a sterile environment.

[0034] In some embodiments, the starting sample is a cell culture. Such a sample may be provided in a bioreactor. In certain embodiments, the bioreactor is a perfusion bioreactor.

[0035] In some embodiments, the capture step may include bind and elute chromatography apparatus that includes at least two separation units, with each unit comprising the same chromatography media, e.g., Protein A affinity media. In a particular embodiment, the Protein A media comprises a Protein A ligand coupled to a rigid hydrophilic polyvinylether polymer matrix. In other embodiments, the Protein A ligand may be coupled to agarose or controlled pore glass. The Protein A ligand may be based on a naturally occurring domain of Protein A from Staphylococcus aureus or be a variant or a fragment of a naturally occurring domain. In a particular embodiment, the Protein A ligand is derived from the C domain of Staphylococcus aureus Protein A. The separation units are connected to be in fluid communication with each other in series, such that a liquid can flow from one separation unit to the next.

[0036] Diafiltration may be used for buffer exchange, desalting and/or sample concentration, such as to remove, replace, or lower the concentration of salts or solvents from solutions containing biomolecules of interest. In certain embodiments, sample may be circulated over an ultrafiltration membrane and returned to the retentate vessel, where fresh buffer is added, while permeate removed.

[0037] In some embodiments, as seen in FIG. 2, upstream of virus filtration, preferably directly upstream thereof, the sample containing the target molecule is subjected to diafiltration, which typically employs the use of an ultrafiltration membrane in a Tangential Flow Filtration (TFF) mode. In case of Tangential Flow Filtration (TFF), the fluid is pumped tangentially along the surface of the filter media and applied pressure serves to force a portion of the fluid through the filter medium to the filtrate side. Diafiltration results in the replacement of the fluid which contains the target molecule with the desired buffer, and allows for proper conditioning or adjustment of the solution conditions, including pH and conductivity. An ultrafilter may be used either in a concentration mode or in a diafiltration model. For batch TFF operation, these can be the same filters run sequentially in different ways. For single pass TFF, these can be sequential filters run in different ways.

[0038] Suitable ultrafiltration membranes for diafiltration include regenerated cellulose and polyethersulfone-based membranes, such as ULTRACEL and BIOMAX membranes commercially available from MilliporeSigma.

[0039] Preferably continuous or constant volume diafiltration is used, where buffer is added at the same rate that filtrate is generated.

[0040] The buffer or buffers chosen for the diafiltration step is preferably the buffer desired for the final formulation of the product, e.g., the drug product. In this way, the subsequent virus filtration step is carried out with the final formulation buffer. Those skilled in the art will know what buffers are suitable for the particular product being manufactured. By way of example, for the drug ERBITUX (cetuximab) for I.V. injection, a suitable buffer is 10 mM citric acid monohydrate. For REOPRO (abciximab), a suitable buffer is 70 mM sodium phosphate.

[0041] After virus filtration in the final formulation buffer, the product can be concentrated as needed such as by ultrafiltration.