Enhanced viral filtration using diafiltration buffer

Abstract

A method and system for purifying a sample containing a biomolecule of interest and impurities includes expressing the biomolecule of interest in a bioreactor to form a product sample containing the biomolecule of interest and impurities; filtering the product sample to form a clarified product sample; subjecting the clarified product sample to affinity chromatography to remove impurities; and then subjecting the product sample to diafiltration, followed by viral filtration and optional concentration. The buffer used during the diafiltration step (and thus in the viral filtration step) is the buffer desired for the final formulation of the product.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application Nos. 62 / 947,082, filed December 12, 2019, and 63 / 049,293, filed July 8, 2020, the entirety of each of which is incorporated herein by reference.

[0002] Field The present disclosure relates to efficient processes and systems for the purification of biomolecules, including therapeutic antibodies and Fc-containing proteins. [Background technology]

[0003] background A general process for producing a biomolecule, such as a protein or virus, particularly a recombinant protein, typically involves two major steps: (1) expression of the protein in a host cell, followed by (2) purification of the protein. The first step involves growing the desired host cell in a bioreactor to induce protein expression. Some examples of cell lines used for this purpose include Chinese hamster ovary (CHO) cells, myeloma (NSO), bacterial cells such as Escherichia coli (e.g., E. coli), and insect cells. Once the protein is expressed at a desired level, it is removed from the host cells and harvested. Suspended particles, such as cells, cell fragments, lipids, and other insoluble materials, are typically removed from the protein-containing solution in a downstream purification process, resulting in a clarified solution containing the protein of interest and other soluble impurities in solution.

[0004] The second step involves purifying the recovered protein to remove impurities inherent in the process. The primary goal of recovery and downstream operations is to isolate the product (e.g., the expressed protein) from soluble and insoluble impurities. Examples of impurities include host cell proteins (HCPs, proteins other than the desired or targeted protein), nucleic acids, endotoxins, viruses, protein variants, protein aggregates, and cell culture media components / additives. This purification typically involves several chromatographic steps, which may include one or more of affinity chromatography, cation exchange chromatography in bind / elute mode, anion exchange chromatography in flow-through mode, hydrophobic interactions on solid matrices such as porous agarose, polymeric or glass, or membrane-based adsorbents.

[0005] One example process template involves a series of chromatography steps, including primary clarification by centrifugation, secondary clarification by filtration, and Protein-A affinity in bind / elute mode, followed by cation exchange in bind / elute mode, followed by anion exchange in flow-through mode. The Protein-A column captures the protein of interest or target protein by affinity mechanisms, while a large amount of impurities pass through the column and are discarded. The protein is then recovered by elution from the column. Because most proteins of interest have isoelectric points (PI) in the basic range (8-9), they are therefore positively charged under normal processing conditions (pH below the protein's PI), and they bind to the cation exchange resin in the second column. Other positively charged impurities also bind to this resin. The protein of interest is then recovered from the column by elution under conditions (pH, salt concentration) that allow the protein to elute but the impurities to remain bound to the resin. Anion exchange columns are typically run in flow-through mode, allowing any negatively charged impurities to bind to the resin, while the positively charged protein of interest is collected in the flow-through stream. After downstream purification processes, viral filtration may be performed, followed by ultrafiltration / diafiltration to adjust the buffer system and concentrate the product, followed by a final packing unit operation to complete the manufacturing process. This process produces a highly pure and concentrated protein solution, which can be particularly important when therapeutic proteins are intended for human use and must be approved by health authorities (e.g., the Food and Drug Administration (FDA)).

[0006] Figure 1 illustrates one conventional process. The process includes a cell harvesting step, which may involve the use of centrifugation to remove cells and cell debris from the cell culture broth, followed by depth filtration. The cell harvesting step is usually followed by a capture step, such as a Protein A affinity purification step, followed by viral inactivation. Viral inactivation is typically followed by one or more chromatography steps (also called polishing steps), which usually include one or more of cation exchange chromatography, anion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, and / or hydroxyapatite chromatography. The polishing step is followed by viral filtration and ultrafiltration / diafiltration, which completes the process.

[0007] The capture step can use a Protein-A resin, such as Eshmuno® A resin (a rigid Protein A affinity chromatography resin) or ProSep® Ultra Plus media (both commercially available from EMD Millipore Corporation, particularly for antibodies containing Fc regions). Other resins operated in bind-and-elute mode can also be suitable for capture. Bind-and-elute chromatography involves (1) loading the product onto the target binding capacity, (2) eluting the product from the column, and (3) washing to prepare the resin for reuse.

[0008] One issue during viral filtration is the L / m 2 During the process of filtering a feed flow rate at a filter throughput of 100 psi, the filter resistance (psi / LMH) increases (or the permeability (LMH / psi) decreases, where LMH is liters / m 2 / h). In other words, the pressure that rises during constant feed flow operation can be compared to the flow rate that falls during constant pressure filtration. These changes are commonly described as filter clogging and are expressed in L / m 2This can significantly limit filter throughput, thereby increasing processing costs ($ / L). Some mechanisms that can cause clogging include retention of high molecular weight (HMW) impurities or retention of aggregates of the therapeutic protein product. As the size of the aggregates increases and approaches the size of viruses, they become retained in the membranes used for virus filtration, where they can deposit on the surface and within the pores of the membrane. This deposition can be associated with a loss of permeability. Proteins can also partially remain in the filter, increasing their internal concentration, viscosity, and osmotic pressure within the filter, creating a clogging effect.

[0009] Size-based membrane filtration provides critical virus removal assurance in most biopharmaceutical molecule production processes. Health authorities expect all therapeutic proteins produced in mammalian viruses to use a virus filtration step to manage the risk of viral contamination in order to grant marketing approval. Many studies have investigated the mechanisms behind the formation of product aggregates, how these aggregates clog virus removal filters, and how filter flow can be maintained by removing the aggregates with adsorptive prefilters. After some throughput, the prefilter may become saturated with aggregates or HMWs, and these species begin to break through and clog the filter. Newer formats (e.g., bispecific antibodies), combined with higher product concentrations associated with enhanced processing, have led to more frequent occurrences of premature filter clogging, despite the implementation of adsorptive prefilters. Summary of the Invention [Problem to be solved by the invention]

[0010] Biotherapeutic formulation is concerned with finding diafiltration buffer solution conditions that promote in-vial stability for extended shelf life and avoid aggregation. Using small-volume analytical techniques for protein aggregation propensity, biotherapeutic formulation chemists rapidly screen multiple buffer systems and excipient concentrations to develop an appropriate diafiltration solution. These objectives mirror the goal of finding virus filter feed solution conditions that avoid virus filter clogging. Therefore, to benefit from the DF buffer stabilization effect and reduced clogging, one may consider moving the location of the virus filtration step from before (i.e., upstream) UF / DF to after (i.e., downstream) UF / DF. This may enable biomolecules such as monoclonal antibodies (Mabs) to be manufactured more efficiently and cost-effectively by operating at higher throughput or higher concentrations. [Means for solving the problem]

[0011] overview The problems of the prior art are overcome by the embodiments disclosed herein, which provide methods and systems for the production of biomolecules. In certain embodiments, virus filtration is performed with the biomolecule in a diafiltration buffer. Preferably, the diafiltration buffer is the same buffer used in the final formulation of the product, ensuring product stability and a long shelf life. A reduction in the virus filtration area can be achieved.

[0012] Embodiments disclosed herein include the purification and isolation of a biomolecule of interest from a cell culture medium. In certain embodiments, the disclosed methods and systems include concentration followed by a downstream purification process. In certain embodiments, the downstream purification process may include sequential purification by one or more chromatography columns.

[0013] In certain embodiments, a method for purifying a sample containing a biomolecule of interest and impurities is disclosed, the method comprising: expressing the biomolecule of interest in a bioreactor to form a product sample containing the biomolecule of interest and impurities; filtering the product sample to clarify the product sample; and subjecting the concentrated product sample to affinity chromatography to remove impurities from the concentrated product sample. In certain embodiments, the product sample (e.g., a harvested cell culture) from the bioreactor is subjected to one or more clarification steps before being subjected to affinity chromatography. The one or more clarification steps may include one or more of centrifugation, tangential flow filtration, depth filtration, and sterile filtration.

[0014] In certain embodiments, the affinity chromatography uses a Protein A affinity ligand.

[0015] In certain embodiments, the method further comprises subjecting the concentrated product sample to a viral inactivation step.

[0016] In certain embodiments, the method includes subjecting the concentrated product sample to a polishing step. In some embodiments, the polishing step includes one or more of anion exchange chromatography, cation exchange chromatography, and hydrophobic interaction chromatography. In some embodiments, both viral inactivation and polishing may be performed.

[0017] In certain embodiments, a diafiltration step is performed prior to virus filtration. In some embodiments, the diafiltration step is performed immediately upstream of the virus filtration, with no intervening unit operations. In various embodiments, the buffer(s) used in the diafiltration step are selected to match the buffer(s) desired for the final product formulation. As a result, the buffer(s) used during the subsequent virus filtration step are the same as the buffer(s) desired for the final formulation.

[0018] In certain embodiments, 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. In some embodiments, the biomolecule is a protein. In other embodiments, the biomolecule is a virus, such as an adeno-associated virus (AAV) vector used for gene therapy. In such cases, viral filtration may be used to pass the AAV product but retain larger exogenous or endogenous viral contaminants.

[0019] In certain embodiments, a system for purifying a biomolecule of interest is disclosed, comprising: a bioreactor; a filter, such as a depth filter, for clarification downstream of the bioreactor; at least two affinity chromatography columns configured in series downstream of the filter; a virus inactivation step or a virus filtration removal step downstream of the at least two affinity chromatography columns; one or more anion exchange, cation exchange, or hydrophobic interaction exchange chromatography columns for purification / polishing, positioned downstream of the virus inactivation step or the virus filtration removal step; one or more ultrafiltration filters configured to operate in a concentration mode or a diafiltration mode, positioned downstream of one or more purification / polishing units (e.g., for batch TFF operation, the same filter can be operated in different ways consecutively, and for single-pass TFF, the series of filters can be operated in different modes); virus filtration downstream of the one or more ultrafiltration filters; and, optionally, an ultrafiltration unit for concentrating the biomolecule of interest downstream of the virus filtration.

[0020] In some embodiments, at least two affinity chromatography columns each contain a Protein A affinity ligand. In some embodiments, there are exactly two affinity chromatography columns. [Brief explanation of the drawings]

[0021] [Figure 1] Schematic of a conventional refining process used in industry. [Figure 2] 1 is a schematic diagram of a purification system according to certain embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description 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 the product of a biomolecule manufacturing process.

[0023] The terms "contaminants," "impurities," and "debris" may be used interchangeably herein and refer to any extraneous or undesirable molecules, including biopolymers such as DNA, RNA, one or more host cell proteins, endotoxins, lipids, protein aggregates, and one or more additives, that may be present in a sample containing a product of interest that is being separated from one or more of the extraneous or undesirable molecules. Additionally, such contaminants may include any reagents used in steps that may occur prior to the separation process.

[0024] As used herein, the term "sample" refers to any composition or mixture containing a target molecule, e.g., 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 comprises a biologic preparation containing the protein of interest to be purified. In particular embodiments, a sample is a cell culture feed containing the protein of interest to be purified. A sample may also comprise diluents, buffers, detergents, and contaminant species, debris, etc., found mixed with the target protein, i.e., the protein of interest. A sample may be "partially purified" (i.e., subjected to one or more purification steps, such as a filtration step), or may be obtained directly from a host cell or organism that produces the target molecule (e.g., a sample may comprise harvested cell culture fluid).

[0025] As used herein, the phrase "consisting essentially of" limits the scope of a claim to certain materials or steps and those that do not materially affect the basic and novel characteristics of the claimed subject matter. This term permits the inclusion of elements and steps that do not materially affect the basic and novel characteristics of the device, system, or method under consideration. Thus, the phrase "consisting essentially of" or "consisting essentially of" means that the recited embodiment, feature, component, step, etc. must be present, and that other embodiments, features, components, steps, etc. may be present so long as their presence does not materially affect the performance, properties, or effects of the recited embodiment, feature, component, step, etc. The presence of operations or steps that have no substantial effect on the sample or product is permitted. For example, a method of purifying a sample comprising a biomolecule of interest and an impurity, consisting essentially of: expressing the biomolecule of interest in a bioreactor to form a product sample comprising the biomolecule of interest and an impurity; filtering the product sample to form a clarified product sample; subjecting the concentrated product sample to affinity chromatography to remove impurities from the concentrated product sample; subjecting the product sample to viral inactivation; subjecting the product sample to purification / polishing, such as by ion exchange chromatography; subjecting the product sample to buffer exchange by diafiltration, and then subjecting the sample to viral filtration, excludes other steps or unit operations that substantially alter the composition of the product sample from being performed between the bioreactor operation and the viral filtration operation, particularly between diafiltration and viral filtration.

[0026] In certain embodiments, the starting sample for the process may vary depending on the cell line in which the sample was grown and the conditions under which the sample was grown and harvested. For example, in most CHO cell processes, cells express molecules in the medium outside the cell wall. Efforts are made to avoid disrupting the cells during harvest to reduce the amount of impurities in the mixture. However, some cells burst or die during growth and harvest due to shear or other operating conditions, becoming lysed and shedding their contents into the mixture. In bacterial cell systems, biomolecules are often protected by or may actually be part of the cell wall (e.g., protein A). In these systems, the cell wall must be disrupted or lysed to recover the biomolecule of interest.

[0027] The target molecule to be purified can be any biomolecule, preferably a protein, in particular a recombinant protein produced in any host cell, including but not limited to Chinese hamster ovary (CHO) cells, the Per.C6® cell line (available from Crucell, 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. Furthermore, the mixture can be a fluid derived from an animal that has been engineered to produce a transgenic fluid (such as milk or blood) containing the biomolecule of interest. Optimal target proteins are antibodies, immunoadhesins and other antibody-like molecules, e.g., C H 2 / C H For example, this product and process may be used for the purification of recombinant humanized monoclonal antibodies, such as (RhuMAb), from harvested conditioned cell culture fluid (HCCF) grown in Chinese hamster ovary (CHO) cells expressing RhuMAb.

[0028] In certain embodiments, a series of purification media with desired chemical functionality are used in a downstream purification process to simultaneously remove soluble impurities while leaving the product in solution and flowing through the purification media to produce a purified stream containing the product. Suitable forms of purification media include derivatized membranes, functionalized chromatography media, or any other porous material with the desired chemical functionality to interact with various impurities, allowing the media to capture impurities through electrostatic, hydrophobic, or affinity interactions. In view of the complex and varied nature of impurities, many purification media with different chemical functionalities may be arranged in series to remove various impurities with different chemical properties.

[0029] In certain embodiments, a process for purifying a target molecule from a sample is disclosed, the process comprising: (a) expressing a protein in a bioreactor to form a protein sample; (b) subjecting the protein sample to filtration, e.g., depth filtration; and (c) subjecting the resulting protein sample to Protein A affinity chromatography using one or more affinity chromatography units.

[0030] Also disclosed is a system for purifying a target molecule from a sample, which includes a bioreactor; a filter unit, e.g., a depth filtration unit; one or more affinity chromatography columns, e.g., one or more Protein A affinity chromatography columns, in fluid communication with the filtration unit; optionally, one or more viral inactivation units downstream of the one or more affinity chromatography columns; optionally, a polishing phase downstream of the affinity chromatography column (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 viral filtration unit; and an ultrafiltration unit downstream of the viral filtration unit.

[0031] In some embodiments, connecting lines exist between the various devices in the system. The devices are connected side by side such that each device in the system is in fluid communication with the device in the preceding or succeeding system.

[0032] In some embodiments, the bioreactor used in the system according to the present invention is a disposable or single-use bioreactor. In some embodiments, the system is enclosed in a sterile environment.

[0033] 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.

[0034] In some embodiments, the capture step may involve a bind-and-elute chromatography device comprising at least two separation units, each unit comprising the same chromatography medium, e.g., Protein A affinity medium. In particular embodiments, the Protein A medium comprises a Protein A ligand bound to a rigid hydrophilic polyvinyl ether polymer matrix. In other embodiments, the Protein A ligand may be bound 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 a variant or fragment of the naturally occurring domain. In certain embodiments, the Protein A ligand is derived from the C domain of Staphylococcus aureus Protein A. The separation units are in series and in fluid communication with one another, allowing liquid to flow from one separation unit to the next.

[0035] Diafiltration may be used for buffer exchange, desalting, and / or sample concentration, e.g., to remove, replace, or reduce the concentration of salts or solvents from a solution containing a biomolecule of interest. In certain embodiments, the sample may be circulated across the ultrafiltration membrane or returned to a retentate vessel, where fresh buffer is added and permeate is simultaneously removed.

[0036] In some embodiments, upstream of, and preferably immediately upstream of, viral filtration, as shown in Figure 2, the sample containing the target molecule is subjected to diafiltration, typically using an ultrafiltration membrane in tangential flow filtration (TFF) mode. In TFF, fluid is drawn tangentially along the surface of the filter medium, and applied pressure forces a portion of the fluid through the filter medium toward the filtrate side. Diafiltration results in the displacement of a fluid containing the target molecule along with a desired buffer, allowing for the correct adjustment or regulation of solution conditions, including pH and conductivity. Ultrafiltration filters can be used in either concentration mode or diafiltration mode. For batch TFF operation, these can be the same filter run sequentially in different ways. For single-pass TFF, these can be multiple filters run sequentially in different ways.

[0037] Suitable ultrafiltration membranes for diafiltration include regenerated cellulose and polyethersulfone-based membranes (eg, ULTRACEL and BIOMAX membranes commercially available from MilliporeSigma).

[0038] Preferably, continuous or constant volume diafiltration is used, where buffer is added at the same rate as filtrate is produced.

[0039] The buffer or buffers selected for the diafiltration step are preferably those desired for the final formulation of the product, e.g., drug product. In this method, the subsequent viral filtration step is performed in the final formulation buffer. One of skill in the art will know what buffer is suitable for the particular product being manufactured. By way of example, a suitable buffer for the IV injection drug ERBITUX (cetuximab) is 10 mM citric acid monohydrate. For REOPRO (abciximab), the buffer is 70 mM sodium phosphate.

[0040] After viral filtration in the final formulation buffer, the product may be concentrated, if necessary, for example by ultrafiltration.

Claims

1. 1. A method for purifying a sample containing a protein of interest and impurities, the method comprising: expressing the protein of interest in a bioreactor to form a product sample containing the protein of interest and impurities; filtering the product sample to form a product sample; subjecting the product sample to affinity chromatography to remove impurities from the product sample; and subjecting the product sample to an initial diafiltration followed immediately by viral filtration, wherein a diafiltration buffer is used in the step of subjecting the product sample to the initial diafiltration, and the product sample is in the same diafiltration buffer when subjected to viral filtration.

2. 10. The method of claim 1, wherein the affinity chromatography comprises a Protein A affinity ligand.

3. 10. The method of claim 1, further comprising subjecting the product sample to a viral inactivation step upstream of the diafiltration.

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

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

6. 2. The method of claim 1, wherein the protein 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. 1. A system for purifying a protein of interest, comprising: a. bioreactor; b. a filtration unit downstream of the bioreactor for clarifying the product sample exiting the 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 unit disposed downstream of said at least two affinity chromatography columns; e. one or more anion exchange, cation exchange, or hydrophobic interaction chromatography columns positioned downstream of the viral inactivation unit; f. a first diafiltration unit disposed downstream of the one or more anion exchange, cation exchange, or hydrophobic interaction chromatography columns, the first diafiltration unit configured to subject a product sample in a diafiltration buffer to diafiltration; and g. a virus filtration unit positioned immediately downstream of said diafiltration unit, said virus filtration unit configured to subject a product sample in said diafiltration buffer to virus filtration. A system comprising:

8. 8. The system of claim 7, wherein the at least two affinity chromatography columns each contain a Protein A affinity ligand.

9. 8. The system of claim 7, configured to operate in batch mode.

10. The system of claim 7 configured to operate in a continuous mode.

11. 1. A system for purifying a protein of interest, comprising: a. bioreactor; b. a filtration unit downstream of the bioreactor for clarifying the product sample exiting the 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 unit disposed downstream of the at least two affinity chromatography membrane units; e. one or more anion exchange, cation exchange or hydrophobic interaction chromatography membrane units positioned downstream of the viral inactivation unit; f. a first diafiltration unit disposed downstream of the one or more anion exchange, cation exchange, or hydrophobic interaction chromatography membrane units, the first diafiltration unit configured to subject a product sample in a diafiltration buffer to diafiltration; and g. a virus filtration unit positioned immediately downstream of said diafiltration unit, said virus filtration unit configured to subject a product sample in said diafiltration buffer to virus filtration. A system comprising:

12. 12. The system of claim 11, wherein the at least two affinity chromatography membrane units each comprise a Protein A affinity ligand.

13. 12. The system of claim 11 configured to operate in batch mode.

14. The system of claim 11 configured to operate in a continuous mode.

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