Methods for chromatography and the reuse of chromatography media

Abstract

This specification discloses methods for chromatography, including a method for washing chromatography media using a linear salt concentration gradient. The disclosed methods may be used for frontal chromatography operations and for washing frontal chromatography media for reuse in the manufacture of biopharmaceuticals.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 509,093, filed June 20, 2023, which is incorporated in its entirety by reference herein.

[0002] This disclosure provides a method for chromatography, including a washing method for a chromatography medium utilizing a linear salt concentration gradient. [Background technology]

[0003] In the manufacture of biopharmaceutical active pharmaceutical ingredients (APIs), high-productivity upstream and downstream operations (i.e., operations capable of processing high-volume APIs) are crucial for large-scale commercial production. API manufacturing operations must consistently deliver high-quality products that meet or exceed the attribute requirements of safety, potency, purity, and efficacy, in quantities sufficient to meet patient demands. In addition, API manufacturing operations must be sustainable, robust, cost-effective, and compliant with regulatory requirements, while maintaining efficient processing within the constraints of manufacturing facility space and time.

[0004] Upstream operations include culturing cells engineered to express the desired product and harvesting the recombinant product. Downstream operations involve purifying the recombinant product from product and process-related impurities and contaminants to meet product quality and yield requirements for the final bulk API. Downstream operations, from capture to polishing, utilize various preparative chromatography methods.

[0005] Contaminants and process-related and product-related impurities are removed to varying degrees by different types of chromatographic media, and downstream purification processes generally use multiple types of chromatographic media to meet product quality requirements. Capture operations, such as affinity chromatography, can remove process-related impurities and contaminants, while polishing chromatography operations can be used to remove product-related impurities and contaminants, such as high molecular weight product-related species. Advances in chromatography technology have made it possible to increase loading density, which can result in chromatographic media becoming saturated with high levels of impurities at the end of a cycle or batch.

[0006] Washing chromatography media is an essential part of clinical and commercial manufacturing processes, affecting product safety, the lifespan of the chromatography media, and the robustness of the process. Washing chromatography media can reduce the possibility of impurities and contaminants being carried over from one cycle to another, as well as fouling, biocontamination, and degradation of the resin in the packed column, thus enabling an increase in the number of lifespan cycles (i.e., the number of times the chromatography media can be recycled without a significant decrease in performance that affects the yield, purity, or other attributes of the purified product).

[0007] Advances in cell line development and culture and harvesting methods have made it possible to obtain high-titer product pools suitable for downstream purification. The increased titer, culture volume, cell density, and impurity levels of products associated with enhanced upstream operations present challenges to downstream operations, including preparative chromatography. To enable efficient downstream purification, additional chromatographic operations capable of achieving high loading densities and robustly separating impurities and contaminants from the desired product over multiple cycles remain necessary in this art. [Overview of the project] [Means for solving the problem]

[0008] Some embodiments of this disclosure describe a method for washing a chromatography medium for reuse, Loading a composition containing protein and at least one impurity onto a chromatography medium, To recover the protein-containing fraction, This includes washing the chromatography medium using a linear salt concentration gradient.

[0009] In some embodiments, the loading density exceeds the dynamic binding capacity of the chromatographic medium to the protein. In some embodiments, the loading density is at least 200 g / Lr. In some embodiments, the loading density is at least 1000 g / Lr. In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr.

[0010] In some embodiments, the chromatography medium is used in frontal mode for protein purification.

[0011] In some embodiments, the chromatography medium is loaded with at least one impurity to a saturated or near-saturated state. In some embodiments, at least one impurity is selected from one or more high molecular weight species of proteins.

[0012] In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from less than 50 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM or 20 mM to 500 mM.

[0013] In some embodiments, the linear salt concentration gradient is an NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient. In some embodiments, the linear salt concentration gradient is an NaCl gradient.

[0014] In some embodiments, a linear salt concentration gradient is generated using at least two buffers. In some embodiments, each of the at least two buffers used to generate the linear salt concentration gradient is independently selected from acetate buffer, phosphate buffer, Tris buffer, and 2-(N-morpholino)ethanesulfonic acid buffer. In some embodiments, each of the at least two buffers used to generate the linear salt concentration gradient contains acetate. In some embodiments, each of the at least two buffers used to generate the linear salt concentration gradient contains acetate at a concentration of 50 mM. In some embodiments, the pH of each of the at least two buffers used to generate the linear salt concentration gradient is greater than 3.6. In some embodiments, the pH of each of the at least two buffers used to generate the linear salt concentration gradient is less than 5.6.

[0015] In some embodiments, a linear salt concentration gradient is generated by buffers A and B, where buffer A contains 50 mM acetate and 0 mM or 20 mM sodium chloride at a pH of 5.0 ± 0.1, and buffer B contains 50 mM acetate and 500 mM sodium chloride at a pH of 5.0 ± 0.1. In some embodiments, a linear salt concentration gradient is generated using buffers A and B in a gradient from 100% buffer A (0% buffer B) to 0% buffer A (100% buffer B), or from 90% buffer A (10% buffer B) to 10% buffer A (90% buffer B), or from 80% buffer A (20% buffer B) to 20% buffer A (80% buffer B).

[0016] In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer solution.

[0017] In some embodiments, the length of the gradient is at least seven times the volume of the medium.

[0018] In some embodiments, washing using a linear salt concentration gradient is non-denaturing.

[0019] In some embodiments, washing using a linear salt concentration gradient is performed after at least 1 cycle. In some embodiments, washing using a linear salt concentration gradient is performed after at least 1 batch. In some embodiments, washing using a linear salt concentration gradient is performed before storage.

[0020] In some embodiments, the method further includes washing the chromatography medium using a denaturing solution. In some embodiments, the base includes sodium hydroxide. In some embodiments, the denaturing solution includes sodium hydroxide at a concentration of 1 M. In some embodiments, 3 times the volume of the medium of the denaturing solution is passed through the chromatography medium.

[0021] In some embodiments, washing using the denaturing solution is performed after at least 1 cycle. In some embodiments, washing using the denaturing solution is performed after at least 1 batch. In some embodiments, washing using the denaturing solution is performed before storage.

[0022] In some embodiments, the denaturing solution is used in a washing process with a uniform concentration.

[0023] In some embodiments, the chromatography medium is stored in a storage solution containing sodium hydroxide at a concentration in the range of 0.1 M to 0.2 M. In some embodiments, the chromatography medium is stored in a storage solution containing sodium hydroxide at a concentration of 0.1 M. In some embodiments, the chromatography medium is stored in a storage solution containing sodium hydroxide at a concentration of 0.2 M.

[0024] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a cilium ligand (e.g., Eshmuno® CP-FT resin).

[0025] In some embodiments, the chromatography medium is packed into the chromatography column.

[0026] In some embodiments, the chromatography medium is a cation exchange chromatography medium and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into the chromatography column.

[0027] In some embodiments, this method allows the chromatography medium to be reused over multiple cycles (e.g., at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles; 5 cycles, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, 50 cycles, 55 cycles, 60 cycles, 65 cycles, 70 cycles, 75 cycles, 80 cycles, 85 cycles, 90 cycles, 95 cycles, 100 cycles). In some embodiments, this method allows the chromatography medium to be reused for at least 10 cycles. In some embodiments, this method allows the chromatography medium to be reused for at least 20 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 30 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 40 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 50 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 60 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 70 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 80 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 90 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 100 cycles.

[0028] In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 200 cm / hour.

[0029] In some embodiments, the linear velocity during loading is 150 cm / hour. In some embodiments, the linear velocity during loading is 200 cm / hour.

[0030] In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 200 cm / hour.

[0031] In some embodiments, the linear velocity during cleaning is 133 cm / hour. In some embodiments, the linear velocity during cleaning is 200 cm / hour.

[0032] Some embodiments of this disclosure describe a method for washing a chromatography medium for reuse, Loading a composition containing a protein and at least one impurity into a chromatographic medium, wherein the loading density exceeds the dynamic binding capacity of the chromatographic medium to the protein, To recover the protein-containing fraction, This includes washing the chromatography medium using a linear salt concentration gradient. A linear salt concentration gradient is a NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient, and A linear salt concentration gradient includes an increase in salt concentration from less than 50 mM to 500 mM.

[0033] In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr.

[0034] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tactile ligand (e.g., Eshmuno® CP-FT resin).

[0035] In some embodiments, the chromatography medium is packed into the chromatography column.

[0036] In some embodiments, the chromatography medium is a cation exchange chromatography medium and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into the chromatography column.

[0037] In some embodiments, the linear salt concentration gradient is a NaCl gradient.

[0038] In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer. In some embodiments, the length of the gradient is at least 7 times the volume of the medium. In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer, and the length of the gradient is at least 7 times the volume of the medium.

[0039] In some embodiments, the linear salt concentration gradient is an NaCl gradient, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer, and the length of the gradient is at least 7 times the volume of the medium.

[0040] In some embodiments, this method allows the chromatography medium to be reused over multiple cycles (e.g., at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles; 5 cycles, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, 50 cycles, 55 cycles, 60 cycles, 65 cycles, 70 cycles, 75 cycles, 80 cycles, 85 cycles, 90 cycles, 95 cycles, 100 cycles). In some embodiments, this method allows the chromatography medium to be reused for at least 10 cycles. In some embodiments, this method allows the chromatography medium to be reused for at least 20 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 30 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 40 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 50 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 60 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 70 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 80 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 90 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 100 cycles.

[0041] In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 200 cm / hour.

[0042] In some embodiments, the linear velocity during loading is 150 cm / hour. In some embodiments, the linear velocity during loading is 200 cm / hour.

[0043] In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 200 cm / hour.

[0044] In some embodiments, the linear velocity during cleaning is 133 cm / hour. In some embodiments, the linear velocity during cleaning is 200 cm / hour.

[0045] Some embodiments of this disclosure describe a method for washing a chromatography medium for reuse, Loading a composition containing a protein and at least one impurity into a chromatographic medium, wherein the loading density exceeds the dynamic binding capacity of the chromatographic medium to the protein, To recover the protein-containing fraction, Washing a chromatography medium using a linear salt concentration gradient, wherein the linear salt concentration gradient is generated by buffer A and buffer B, where buffer A contains 50 mM acetate and 0 mM or 20 mM sodium chloride at a pH of 5.0 ± 0.1, and buffer B contains 50 mM acetate and 500 mM sodium chloride at a pH of 5.0 ± 0.1.

[0046] In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr.

[0047] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tactile ligand (e.g., Eshmuno® CP-FT resin).

[0048] In some embodiments, the chromatography medium is packed into the chromatography column.

[0049] In some embodiments, the chromatography medium is a cation exchange chromatography medium and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into the chromatography column.

[0050] In some embodiments, a linear salt concentration gradient is generated using buffers A and B with a gradient from 100% buffer A (0% buffer B) to 0% buffer A (100% buffer B), or from 90% buffer A (10% buffer B) to 10% buffer A (90% buffer B), or from 80% buffer A (20% buffer B) to 20% buffer A (80% buffer B).

[0051] In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer. In some embodiments, the length of the gradient is at least 7 times the volume of the medium. In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume of buffer, and the length of the gradient is at least 7 times the volume of the medium.

[0052] In some embodiments, this method allows the chromatography medium to be reused over multiple cycles (e.g., at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles; 5 cycles, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, 50 cycles, 55 cycles, 60 cycles, 65 cycles, 70 cycles, 75 cycles, 80 cycles, 85 cycles, 90 cycles, 95 cycles, 100 cycles). In some embodiments, this method allows the chromatography medium to be reused for at least 10 cycles. In some embodiments, this method allows the chromatography medium to be reused for at least 20 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 30 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 40 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 50 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 60 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 70 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 80 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 90 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 100 cycles.

[0053] In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 200 cm / hour.

[0054] In some embodiments, the linear velocity during loading is 150 cm / hour. In some embodiments, the linear velocity during loading is 200 cm / hour.

[0055] In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 200 cm / hour.

[0056] In some embodiments, the linear velocity during cleaning is 133 cm / hour. In some embodiments, the linear velocity during cleaning is 200 cm / hour.

[0057] Some embodiments of this disclosure describe a method for washing a chromatography medium for reuse, Loading a composition containing a protein and at least one impurity into a chromatographic medium, wherein the loading density exceeds the dynamic binding capacity of the chromatographic medium to the protein, To recover the protein-containing fraction, The method involves washing the chromatography medium using a linear salt concentration gradient, wherein the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / medium volume buffer.

[0058] In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr.

[0059] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tactile ligand (e.g., Eshmuno® CP-FT resin).

[0060] In some embodiments, the chromatography medium is packed into the chromatography column.

[0061] In some embodiments, the chromatography medium is a cation exchange chromatography medium and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into the chromatography column.

[0062] In some embodiments, the linear salt concentration gradient is an NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient. In some embodiments, the linear salt concentration gradient is an NaCl gradient.

[0063] In some embodiments, the length of the gradient is at least seven times the volume of the medium.

[0064] In some embodiments, the linear salt concentration gradient is an NaCl gradient, and the length of the gradient is at least 7 times the volume of the medium.

[0065] In some embodiments, this method allows the chromatography medium to be reused over multiple cycles (e.g., at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles; 5 cycles, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, 50 cycles, 55 cycles, 60 cycles, 65 cycles, 70 cycles, 75 cycles, 80 cycles, 85 cycles, 90 cycles, 95 cycles, 100 cycles). In some embodiments, this method allows the chromatography medium to be reused for at least 10 cycles. In some embodiments, this method allows the chromatography medium to be reused for at least 20 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 30 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 40 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 50 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 60 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 70 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 80 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 90 cycles. In some embodiments, the method allows for the reuse of the chromatography medium for at least 100 cycles.

[0066] In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during loading is in the range of 150 cm / hour to 200 cm / hour.

[0067] In some embodiments, the linear velocity during loading is 150 cm / hour. In some embodiments, the linear velocity during loading is 200 cm / hour.

[0068] In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 100 cm / hour to 200 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 250 cm / hour. In some embodiments, the linear velocity during cleaning is in the range of 125 cm / hour to 200 cm / hour.

[0069] In some embodiments, the linear velocity during cleaning is 133 cm / hour. In some embodiments, the linear velocity during cleaning is 200 cm / hour.

[0070] Some embodiments of this disclosure relate to a method for controlling the operating pressure during washing of a chromatography medium for reuse. Washing the chromatography medium using a linear salt concentration gradient, This includes washing the chromatography medium with a denatured solution.

[0071] In some embodiments, the modified solution is used in a washing process with a uniform concentration.

[0072] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tactile ligand (e.g., Eshmuno® CP-FT resin).

[0073] In some embodiments, the chromatography medium is packed into the chromatography column.

[0074] In some embodiments, the chromatography medium is a cation exchange chromatography medium and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into the chromatography column.

[0075] In some embodiments, this method allows the chromatography medium to be reused over multiple cycles (e.g., at least 5 cycles, at least 10 cycles, at least 15 cycles, at least 20 cycles, at least 25 cycles, at least 30 cycles, at least 35 cycles, at least 40 cycles, at least 45 cycles, at least 50 cycles, at least 55 cycles, at least 60 cycles, at least 65 cycles, at least 70 cycles, at least 75 cycles, at least 80 cycles, at least 85 cycles, at least 90 cycles, at least 95 cycles, at least 100 cycles; 5 cycles, 10 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, 50 cycles, 55 cycles, 60 cycles, 65 cycles, 70 cycles, 75 cycles, 80 cycles, 85 cycles, 90 cycles, 95 cycles, 100 cycles). In some embodiments, this method allows the chromatography medium to be reused for at least 10 cycles. In some embodiments, this method allows the chromatography medium to be reused for at least 20 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 30 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 40 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 50 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 60 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 70 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 80 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 90 cycles. In some embodiments, this method allows for the reuse of the chromatography medium for at least 100 cycles.

[0076] Some embodiments of this disclosure are methods for purifying a protein from a composition comprising a protein and at least one impurity, Equilibrating the chromatography medium using an equilibration solution, A composition containing protein and at least one impurity is loaded onto a chromatography medium in frontal mode at a loading density of at least 500 g / Lr. To recover the protein-containing fraction, The loading rinse solution, in an amount less than twice the volume of the medium, is passed through the chromatography medium, and the eluate is added to the fraction. Washing the chromatography medium using a linear salt concentration gradient, This includes washing the chromatography medium with a denatured solution.

[0077] In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr.

[0078] In some embodiments, fraction recovery is initiated at an optical concentration (OD) of 0.5 at A280 (absorbance at 280 nm). In some embodiments, fraction recovery is stopped after passing a post-loading rinse solution (less than one-fold volume of the medium) through the chromatographic medium.

[0079] In some embodiments, the equilibration solution does not contain salt.

[0080] In some embodiments, the modified solution is used in a washing process with a uniform concentration.

[0081] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography resin.

[0082] In some embodiments, the chromatography medium is packed into the chromatography column.

[0083] In some embodiments, the chromatography medium is a cation exchange chromatography medium, which is packed into the chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin, which is packed into the chromatography column.

[0084] Some embodiments of this disclosure are methods for purifying a protein from a composition comprising a protein and at least one impurity, Initiating cell culture in a bioreactor, Culturing cells that have been manipulated to express proteins, Harvesting cell culture broth containing protein, The harvested cell culture broth is loaded onto an affinity chromatography medium in binding and elution mode, To recover the affinity chromatography elution fraction containing protein, The affinity chromatography elution fraction is loaded onto a cation exchange (CEX) chromatography medium in frontal mode at a loading density of at least 500 g / Lr. To recover the CEX chromatography fraction containing protein, Washing the CEX chromatography medium using a linear salt concentration gradient, The present invention relates to a method comprising washing a CEX chromatography medium with a denaturing solution.

[0085] In some embodiments, the affinity chromatography medium is a protein A affinity chromatography medium.

[0086] In some embodiments, the affinity chromatography eluted fraction is subjected to low-pH viral inactivation. In some embodiments, the affinity chromatography eluted fraction is subjected to low-pH viral inactivation and neutralization after viral inactivation. In some embodiments, the neutralization pH is at least 5.0 (e.g., 5.0). In some embodiments, the protein is eluted from the affinity chromatography medium at a pH suitable for viral inactivation. In some embodiments, the protein is eluted from the affinity chromatography medium at a pH of 4.0 or lower. In some embodiments, the pH of the eluted protein is adjusted to at least pH 5.0 (e.g., 5.0).

[0087] In some embodiments, the affinity chromatography elution fraction is loaded onto the CEX chromatography medium for a loading time sufficient to achieve viral inactivation. In some embodiments, this loading time is at least 30 minutes.

[0088] In some embodiments, the chromatography medium is a cation exchange chromatography resin. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin). In some embodiments, the CEX chromatography medium is packed into a chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin and is packed into a chromatography column. In some embodiments, the chromatography medium is a cation exchange chromatography resin containing a tentacle ligand (e.g., Eshmuno® CP-FT resin) and is packed into a chromatography column.

[0089] Some embodiments of the present disclosure relate to a method for improving step yield for a frontal chromatography operation, the method comprising estimating the amount of protein in a loading solution and determining the bed height of the frontal chromatography medium based on a target residence time, wherein the flow rate is a constant value in the range of 50 cm / hour to 250 cm / hour (e.g., 50 cm / hour, 75 cm / hour, 100 cm / hour, 125 cm / hour, 150 cm / hour, 175 cm / hour, 200 cm / hour, 225 cm / hour, 250 cm / hour, etc.).

[0090] In some embodiments, the flow velocity is in the range of 100 cm / hour to 250 cm / hour.

[0091] In some embodiments, the flow velocity is in the range of 100 cm / hour to 200 cm / hour.

[0092] In some embodiments, the flow velocity is 133 cm / hour or 200 cm / hour.

[0093] In some embodiments, the bed diameter of the frontal chromatography medium is kept constant.

[0094] In some embodiments, the bed diameter is greater than 1 cm, greater than 2 cm, greater than 3 cm, greater than 4 cm, greater than 5 cm, greater than 10 cm, greater than 15 cm, greater than 20 cm, greater than 25 cm, greater than 30 cm, greater than 35 cm, greater than 40 cm, or greater than 45 cm. In some embodiments of the methods described herein, the bed diameter is greater than 80 cm, greater than 100 cm, or greater than 120 cm.

[0095] In some embodiments, the bed diameter is greater than 1 cm. In some embodiments, the bed diameter is greater than 10 cm.

[0096] In some embodiments, the bed diameter is 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, or 45 cm. In some embodiments, the bed diameter is 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 100 cm, 110 cm, 120 cm, 130 cm, 140 cm, 150 cm, 160 cm, 170 cm, 180 cm, 190 cm, or 200 cm.

[0097] In some embodiments, the bed diameter is in the range of 1 cm to 20 cm. In some embodiments, the bed diameter is 1 cm, 10 cm, or 20 cm. In some embodiments, the bed diameter is 1 cm. In some embodiments, the bed diameter is 10 cm. In some embodiments, the bed diameter is 20 cm.

[0098] In some embodiments, the bed height is a constant value in the range of 5 cm to 30 cm. In some embodiments, the bed height is a constant value in the range of 10 cm to 20 cm.

[0099] In some embodiments, the amount of protein in the loading solution is estimated based on monitoring performed in real time, near real time, and / or offline. In some embodiments, the amount of protein in the loading solution is estimated based on historical data. In some embodiments, the viral prefilter is replaced after one or more filtration cycles. In some embodiments, estimation is performed after one or more batches.

[0100] In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin containing tentacle ligands (e.g., Eshmuno® CP-FT resin). In some embodiments, the frontal chromatography medium is packed into a chromatography column. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin and is packed into a chromatography column. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin containing tentacle ligands (e.g., Eshmuno® CP-FT resin) and is packed into a chromatography column.

[0101] Some embodiments of the present disclosure relate to a method for improving step yield for a frontal chromatography operation, the method comprising estimating the amount of protein in the loading solution and determining the flow rate of the frontal chromatography operation based on a target residence time, wherein the flow rate is in the range of 50 cm / hour to 250 cm / hour and the bed height of the frontal chromatography medium is constant in the range of 5 cm to 30 cm.

[0102] In some embodiments, the bed diameter of the frontal chromatography medium is kept constant.

[0103] In some embodiments, the bed diameter is greater than 1 cm, greater than 2 cm, greater than 3 cm, greater than 4 cm, greater than 5 cm, greater than 10 cm, greater than 15 cm, greater than 20 cm, greater than 25 cm, greater than 30 cm, greater than 35 cm, greater than 40 cm, or greater than 45 cm. In some embodiments of the methods described herein, the bed diameter is greater than 80 cm, greater than 100 cm, or greater than 120 cm.

[0104] In some embodiments, the bed diameter is greater than 1 cm. In some embodiments, the bed diameter is greater than 10 cm.

[0105] In some embodiments, the bed diameter is 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, or 45 cm. In some embodiments, the bed diameter is 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 100 cm, 110 cm, 120 cm, 130 cm, 140 cm, 150 cm, 160 cm, 170 cm, 180 cm, 190 cm, or 200 cm.

[0106] In some embodiments, the bed diameter is in the range of 1 cm to 20 cm. In some embodiments, the bed diameter is 1 cm, 10 cm, or 20 cm. In some embodiments, the bed diameter is 1 cm. In some embodiments, the bed diameter is 10 cm. In some embodiments, the bed diameter is 20 cm.

[0107] In some embodiments, the flow velocity is in the range of 100 cm / hour to 250 cm / hour. In some embodiments, the flow velocity is in the range of 100 cm / hour to 200 cm / hour.

[0108] In some embodiments, the flow velocity is 133 cm / hour or 200 cm / hour.

[0109] In some embodiments, the bed height is a constant value within the range of 10 cm to 20 cm.

[0110] In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin containing tentacle ligands (e.g., Eshmuno® CP-FT resin). In some embodiments, the frontal chromatography medium is packed into a chromatography column. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin and is packed into a chromatography column. In some embodiments, the frontal chromatography medium is a cation exchange chromatography resin containing tentacle ligands (e.g., Eshmuno® CP-FT resin) and is packed into a chromatography column.

[0111] In some embodiments, the amount of protein in the loading solution is estimated based on monitoring performed in real time, near real time, and / or offline. In some embodiments, the amount of protein in the loading solution is estimated based on historical data. In some embodiments, the viral prefilter is replaced after one or more filtration cycles. In some embodiments, estimation is performed after one or more batches. [Brief explanation of the drawing]

[0112] [Figure 1] This is a schematic diagram showing the basic operation sequence from equilibration to storage of Frontal CEX chromatography resin. [Figure 2] This graph shows the maximum ΔP (change in column pressure) observed during uniform concentration strip cleaning (50 mM acetate, 1 M CaCl2, pH 5.0) of a Frontal CEX chromatography column used to purify mAbs, with a column loading volume of 750 g / Lr, pH 5.0, and a loading material with a conductivity of 5.3 mS / cm. [Figure 3]This document shows the ΔP profiles and UV absorbance (A280, absorbance at 280 nm) of bench-scale equilibration, frontal loading, rinsing, unmodified strip, and modified strip of CEX resin washed using an unmodified linear salt concentration gradient. Specifically, after rinsing, the resin was subjected to an unmodified washing step consisting of a linear salt concentration gradient of 7 CV or more (0-100% buffer B (Buffer A: 50 mM acetate, 0 M NaCl, pH 5.0; Buffer B: 50 mM acetate, 0.5 M NaCl, pH 5.0)), followed by a modification step using 1 M NaOH. The resin was then stored in 0.2 M NaOH. Absorbance (280 nM) is shown by the thin line. ΔP is shown by the thick line. [Figure 4] This shows the inter-cycle pre-column pressure using linear salt concentration gradient washing over multiple cycles (100 cycles). [Figure 5] The step yield as a function of column loading volume for the Frontal CEX chromatography step used to purify mAbs is shown. The data includes both pilot-scale and bench-scale chromatography operations, and includes loading material for bench-scale data obtained from pilot-scale bioreactors (500 L–2000 L). [Figure 6] This graph shows the predicted step yield of the frontal CEX step performed in four model facilities using different target bed heights under different facility constraints. [Modes for carrying out the invention]

[0113] In biopharmaceutical manufacturing operations, polishing chromatography can be used to remove a variety of product and process-related impurities, including high molecular weight (HMW) and low molecular weight (LMW) product-related species. For example, cation exchange (CEX) chromatography operated in binding and elution modes is widely used due to its high chromatographic resolution for the product species of interest, robust clearance of host cell impurities and viruses, and reasonably high yields. However, the removal of significant impurities, such as HMW product-related species, generally decreases with increasing loading density. The loading capacity of a polishing chromatography operation can constrain the overall throughput of the downstream process. In such cases, strategies for processing entire product batches within the facility's target utilization rate may require scaling up the diameter of the rate-limited chromatography operation and / or parallel processing of lots in multiple columns, both of which are expensive mitigations that may not be feasible within the facility's footprint and operational parameters. In addition, these strategies may increase buffer volume requirements along with energy and water consumption, and may reduce flexibility for adapting to improvements in upstream titer and / or productivity.

[0114] In binding and elution modes, a chromatographic medium having some type of affinity for the target product is used. The product binds to the chromatographic medium during loading. The bound product is then eluted and recovered as a pool of products. Certain impurities, such as product-related species, may bind to the chromatographic medium during loading, depending on whether they have a higher or lower affinity than the target product. Various strategies are employed to purify the desired product from co-bound impurities, including the use of pre-elution rinses to desorb undesirable low-affinity impurities, gradient elution which gradually changes the composition of the mobile phase to improve the separation between the target product and impurities during elution, and the design of pool collection criteria to collect the target product without introducing low-affinity or high-affinity impurities.

[0115] To achieve increased loading density during the polishing chromatography step, alternative chromatographic operating modes such as flow-through chromatography, weak partition chromatography, overload chromatography, and frontal loading chromatography may be used. In flow-through mode, the chromatographic medium and operating conditions are favorable for binding impurities and contaminants as the desired product flows, and the effluent concentrates the desired product. In overload chromatography, the product is loaded onto the chromatographic material beyond its dynamic binding capacity. Both weak partition and frontal loading chromatography modes separate the desired product from impurities and contaminants by relying on competitive binding of components to the chromatographic medium during loading. For example, frontal loading mode is characterized by continuous loading under conditions where all components in the loading feed first bind to the chromatographic medium. In an ideal operation, as loading progresses, the binding components are replaced in order of their affinity to the chromatographic medium (i.e., stationary phase) until the medium is largely saturated with components of high binding affinity. As an example, in cation exchange chromatography (CEX) operations using a frontal loading mode designed to bind positively charged molecular species, the target product (i.e., monomers) and low molecular weight (LMW) product-related impurities (i.e., cleaved proteins expressed during culture, degraded proteins, and enzymatically cleaved proteins) are typically not retained very strongly and are rapidly replaced by more positively charged components. The target product and LMW impurities flow into the effluent, which is either carried downstream to the unit operation or recovered as a product pool. Chromatographic operations using the frontal loading mode can remove HMW product-related species such as dimers, oligomers, and higher-order aggregates at high loading densities, as well as process-related impurities with higher binding affinity and retention in the chromatography medium, such as host cell proteins (HCPs).As loading progresses, the chromatographic medium becomes saturated with more affinity impurities, and the effluent becomes concentrated with the desired product. Beyond this saturation point, additional impurities loaded into the resin are expected to flow into the effluent along with the desired product. Therefore, the saturation point of a critical impurity designed to bind during a frontal loading chromatography step (e.g., HMW product-related impurities) typically represents a practical upper limit of the loading density.

[0116] High loading of the chromatography medium in frontal loading mode can provide HMW impurity removal that is more than an order of magnitude higher than loading in binding and elution modes. Higher loading rates may be desirable to improve the utilization rate of the chromatography medium (e.g., per unit volume of loading chromatography column), reduce buffer consumption, and increase productivity. The degree of these improvements depends on process parameters such as the allowable loading density, operating flow rate, bed height, and feed flow concentration.

[0117] However, one limitation of frontal chromatography compared to binding and elution chromatography is the high correlation between the loading amount and yield in the frontal step. Increased loading beyond saturation with the desired product increases the proportion of unbound loading product, thus increasing the yield of recovered product from the frontal chromatography operation. Conversely, the relative amount of product remaining bound to the resin decreases, resulting in a non-linear relationship between yield and loading. Thus, the overall process yield is sensitive to factors affecting the amount of protein available for loading, such as changes in the titer of the product in the loading buffer.

[0118] Another limitation of frontal chromatography is the challenge posed by high loading for chromatographic reuse. Chromatographic reuse involves washing and / or regenerating the chromatographic medium and using it for the purification of the same or different products. High loading densities can lead to saturation or near-saturation of the chromatographic medium with impurities such as host cell proteins, potentially resulting in high column pressure during the regeneration / rinsing and / or sterilization stages of the chromatographic medium. Insufficient washing and / or high column pressure can reduce the number of reuse cycles for the chromatographic medium and affect performance in subsequent cycles.

[0119] Non-limiting embodiment Non-limiting embodiments of this disclosure include, but are not limited to, the following:

[0120] Embodiment 1. A method for washing chromatography media for reuse, Loading a composition containing protein and at least one impurity onto a chromatography medium, To recover the protein-containing fraction, A method comprising washing a chromatography medium using a linear salt concentration gradient.

[0121] Embodiment 2. The method according to Embodiment 1, wherein the loading density exceeds the dynamic binding capacity of the chromatography medium to the protein.

[0122] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the loading density is at least 200 g / Lr.

[0123] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the loading density is at least 1000 g / Lr (for example, in the range of 1000 g / Lr to 1500 g / Lr).

[0124] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the chromatography medium is used in frontal mode for protein purification.

[0125] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the chromatography medium is loaded with at least one impurity in a saturated or nearly saturated state.

[0126] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein at least one impurity comprises at least one high molecular weight species of protein.

[0127] Embodiment 8. The method according to any one of Embodiments 1 to 7, wherein the linear salt concentration gradient includes an increase in salt concentration from less than 50 mM to 500 mM.

[0128] Embodiment 9. The method according to any one of Embodiments 1 to 7, wherein the linear salt concentration gradient includes an increase in salt concentration from 0 mM or 20 mM to 500 mM.

[0129] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the linear salt concentration gradient is an NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient.

[0130] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the linear salt concentration gradient is an NaCl gradient.

[0131] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein a linear salt concentration gradient is generated using at least two buffer solutions.

[0132] Embodiment 13. The method according to Embodiment 12, wherein each of the at least two buffers used to generate a linear salt concentration gradient is independently selected from acetate buffer, phosphate buffer, Tris buffer, and 2-(N-morpholino)ethanesulfonic acid buffer.

[0133] Embodiment 14. The method according to Embodiment 12 or Embodiment 13, wherein each of the at least two buffer solutions used to generate a linear salt concentration gradient contains an acetate.

[0134] Embodiment 15. The method according to any one of Embodiments 12 to 14, wherein each of the at least two buffer solutions used to generate a linear salt concentration gradient contains an acetate at a concentration of 50 mM.

[0135] Embodiment 16. The method according to any one of Embodiments 12 to 15, wherein the pH of each of the at least two buffers used to generate a linear salt concentration gradient is greater than 3.6.

[0136] Embodiment 17. The method according to any one of Embodiments 1 to 16, wherein the pH of each of the at least two buffers used to generate a linear salt concentration gradient is less than 5.6.

[0137] Embodiment 18. The method according to any one of Embodiments 1 to 11, wherein a linear salt concentration gradient is generated by buffer A and buffer B, wherein buffer A has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 0 mM or 20 mM sodium chloride, and buffer B has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 500 mM sodium chloride.

[0138] Embodiment 19. The method according to Embodiment 18, wherein a linear salt concentration gradient is generated using buffers A and B with a gradient from 100% buffer A (0% buffer B) to 0% buffer A (100% buffer B), or from 90% buffer A (10% buffer B) to 10% buffer A (90% buffer B), or from 80% buffer A (20% buffer B) to 20% buffer A (80% buffer B).

[0139] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / medium volume of buffer solution.

[0140] Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein the length of the gradient is at least 7 times the volume of the medium.

[0141] Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the washing using a linear salt concentration gradient is non-denaturing.

[0142] Embodiment 23. The method according to any one of Embodiments 1 to 22, wherein washing using a linear salt concentration gradient is performed at least once after one cycle.

[0143] Embodiment 24. The method according to any one of Embodiments 1 to 22, wherein washing using a linear salt concentration gradient is performed at least one batch later.

[0144] Embodiment 25. The method according to any one of Embodiments 1 to 22, wherein washing using a linear salt concentration gradient is performed before storage.

[0145] Embodiment 26. The method according to any one of Embodiments 1 to 25, further comprising washing the chromatography medium using a uniform concentration washing process using a denatured solution.

[0146] Embodiment 27. The method according to Embodiment 26, wherein the denatured solution contains sodium hydroxide.

[0147] Embodiment 28. The method according to Embodiment 26 or Embodiment 27, wherein the denatured solution contains sodium hydroxide at a concentration of 1 M.

[0148] Embodiment 29. The method according to any one of Embodiments 26 to 28, wherein a denatured solution in an amount three times the volume of the medium is passed through the chromatographic medium.

[0149] Embodiment 30. The method according to any one of Embodiments 26 to 29, wherein washing using a denaturing solution is performed at least once after one cycle.

[0150] Embodiment 31. The method according to any one of Embodiments 26 to 29, wherein washing using a denaturing solution is performed after at least one batch.

[0151] Embodiment 32. The method according to any one of Embodiments 26 to 29, wherein washing using a denaturing solution is performed before storage.

[0152] Embodiment 33. The method according to Embodiment 25 or Embodiment 32, wherein the chromatography medium is stored in a storage solution containing sodium hydroxide at a concentration in the range of 0.1 M to 0.2 M.

[0153] Embodiment 34. A method for controlling the operating pressure during washing of a chromatography medium for reuse, Washing the chromatography medium using a linear salt concentration gradient, This includes washing the chromatography medium with a denatured solution.

[0154] Embodiment 35. A method for purifying a protein from a composition containing a protein and at least one impurity, Equilibrating the chromatography medium using equilibration buffer, A composition containing protein and at least one impurity is loaded onto a chromatography medium in frontal mode at a loading density of at least 500 g / Lr. To recover the protein-containing fraction, The loading rinse solution, in an amount less than twice the volume of the medium, is passed through the chromatography medium, and the eluate is added to the fraction. Washing the chromatography medium using a linear salt concentration gradient, This includes washing the chromatography medium with a denatured solution.

[0155] Embodiment 36. The method according to Embodiment 35, wherein fraction recovery is started at an OD of 0.5 at A280 (absorbance at 280 nm).

[0156] Embodiment 37. The method according to Embodiment 35 or Embodiment 36, wherein fraction recovery is stopped after passing a rinse solution through the chromatography medium after loading an amount equal to or less than one times the volume of the medium.

[0157] Embodiment 38. The method according to any one of Embodiments 35 to 37, wherein the equilibration buffer does not contain salt.

[0158] Embodiment 39. A method for purifying a protein from a composition containing a protein and at least one impurity, Initiating cell culture in a bioreactor, Culturing cells that have been manipulated to express proteins, Harvesting cell culture broth containing protein, The harvested cell culture broth is loaded onto an affinity chromatography medium in binding and elution mode, To recover the affinity chromatography elution fraction containing protein, The affinity chromatography elution fraction is loaded onto a cation exchange (CEX) chromatography medium in frontal mode at a loading density of at least 500 g / Lr. To recover the CEX chromatography fraction containing protein, Washing the CEX chromatography medium using a linear salt concentration gradient, A method comprising washing a CEX chromatography medium with a denaturing solution.

[0159] Embodiment 40. The method according to Embodiment 39, wherein the affinity chromatography medium is a protein A affinity chromatography medium.

[0160] Embodiment 41. The method according to Embodiment 39 or Embodiment 40, wherein the affinity chromatography elution fraction is subjected to low pH virus inactivation and optional neutralization after virus inactivation.

[0161] Embodiment 42. The method according to any one of Embodiments 39 to 41, wherein the protein is eluted from the affinity chromatography medium at a pH suitable for virus inactivation.

[0162] Embodiment 43. The method according to any one of Embodiments 39 to 42, wherein the protein is eluted from the affinity chromatography medium at a pH of 4.0 or lower.

[0163] Embodiment 44. The method according to Embodiment 42 or Embodiment 43, wherein the affinity chromatography elution fraction is loaded onto a CEX chromatography medium for a loading time sufficient to achieve virus inactivation.

[0164] Embodiment 45. The method according to Embodiment 44, wherein the loading time is at least 30 minutes.

[0165] Embodiment 46. The method according to any one of Embodiments 34 to 45, wherein the denatured solution is used in a washing process at a uniform concentration.

[0166] Embodiment 47. The method according to any one of Embodiments 1 to 38, wherein the chromatography medium is a cation exchange (CEX) chromatography medium.

[0167] Embodiment 48. The method according to any one of Embodiments 1 to 47, wherein the chromatography medium is packed into the chromatography column.

[0168] Embodiment 49. A method for washing a chromatography medium for reuse, Loading a composition containing a protein and at least one impurity into a chromatographic medium, wherein the loading density exceeds the dynamic binding capacity of the chromatographic medium to the protein, To recover the protein-containing fraction, A method comprising washing a chromatography medium using a linear salt concentration gradient.

[0169] Embodiment 50. The method according to Embodiment 49, wherein the chromatography medium is a cation exchange (CEX) chromatography resin.

[0170] Embodiment 51. The method according to Embodiment 49 or Embodiment 50, wherein the chromatography medium is packed into the chromatography column.

[0171] Embodiment 52. The method according to any one of Embodiments 49 to 51, wherein the loading density is at least 200 g / Lr.

[0172] Embodiment 53. The method according to any one of Embodiments 49 to 52, wherein the loading density is at least 1000 g / Lr (for example, in the range of 1000 g / Lr to 1500 g / Lr).

[0173] Embodiment 54. The method according to any one of Embodiments 49 to 53, wherein the chromatography medium is loaded with at least one impurity in a saturated or nearly saturated state.

[0174] Embodiment 55. The method according to any one of Embodiments 49 to 54, wherein at least one impurity comprises at least one high molecular weight species of protein.

[0175] Embodiment 56. The method according to any one of Embodiments 49 to 55, wherein the linear salt concentration gradient includes an increase in salt concentration from less than 50 mM to 500 mM.

[0176] Embodiment 57. The method according to any one of Embodiments 49 to 56, wherein the linear salt concentration gradient is an NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient.

[0177] Embodiment 58. The method according to any one of Embodiments 49 to 57, wherein the linear salt concentration gradient is an NaCl gradient.

[0178] Embodiment 59. The method according to any one of Embodiments 49 to 58, wherein a linear salt concentration gradient is generated using at least two buffers, and each of the at least two buffers used to generate the linear salt concentration gradient is independently selected from acetate buffer, phosphate buffer, Tris buffer, and 2-(N-morpholino)ethanesulfonic acid buffer.

[0179] Embodiment 60. The method according to Embodiment 59, wherein each of the at least two buffer solutions used to generate a linear salt concentration gradient contains an acetate.

[0180] Embodiment 61. The method according to Embodiment 59 or Embodiment 60, wherein the pH of each of the at least two buffers used to generate a linear salt concentration gradient is greater than 3.6 and less than 5.6.

[0181] Embodiment 62. The method according to any one of Embodiments 49 to 58, wherein a linear salt concentration gradient is generated by buffer A and buffer B, wherein buffer A has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 0 mM or 20 mM sodium chloride, and buffer B has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 500 mM sodium chloride.

[0182] Embodiment 63. The method according to Embodiment 62, wherein a linear salt concentration gradient is generated using buffers A and B with a gradient from 100% buffer A (0% buffer B) to 0% buffer A (100% buffer B), or from 90% buffer A (10% buffer B) to 10% buffer A (90% buffer B), or from 80% buffer A (20% buffer B) to 20% buffer A (80% buffer B).

[0183] Embodiment 64. The method according to any one of Embodiments 49 to 63, wherein the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / medium volume of buffer solution.

[0184] Embodiment 65. The method according to any one of Embodiments 49 to 64, wherein the length of the gradient is at least 7 times the volume of the medium.

[0185] Embodiment 66. The method according to any one of Embodiments 49 to 65, wherein the washing using a linear salt concentration gradient is non-denaturing.

[0186] Embodiment 67. The method according to any one of Embodiments 49 to 66, further comprising washing the chromatography medium using a uniform concentration washing process using a denatured solution.

[0187] Embodiment 68. The method according to Embodiment 67, wherein the denatured solution contains sodium hydroxide.

[0188] Embodiment 69. The method according to any one of Embodiments 49 to 68, which enables the reuse of a chromatography medium for multiple cycles.

[0189] Embodiment 70. The method according to any one of Embodiments 49 to 68, which enables the reuse of the chromatography medium for at least 25 cycles.

[0190] Embodiment 71. A method for purifying a protein from a composition containing a protein and at least one impurity, Equilibrating the chromatography medium using equilibration buffer, A composition containing protein and at least one impurity is loaded onto a chromatography medium in frontal mode at a loading density of at least 500 g / Lr. To recover the protein-containing fraction, The loading rinse solution, in an amount less than twice the volume of the medium, is passed through the chromatography medium, and the eluate is added to the fraction. Washing the chromatography medium using a linear salt concentration gradient, This includes washing the chromatography medium with a denatured solution.

[0191] Embodiment 72. The method according to Embodiment 71, wherein fraction recovery is started at an OD of 0.5 at A280 (absorbance at 280 nm).

[0192] Embodiment 73. The method according to Embodiment 71 or Embodiment 72, wherein fraction recovery is stopped after passing a rinse solution through a chromatography medium after loading an amount equal to or less than one times the volume of the medium.

[0193] Embodiment 74. The method according to any one of Embodiments 71 to 73, wherein the equilibration buffer does not contain salt.

[0194] Embodiment 75. The method according to any one of Embodiments 71 to 74, wherein the denatured solution is used in a washing process at a uniform concentration.

[0195] Embodiment 76. The method according to any one of Embodiments 71 to 75, wherein the chromatography medium is a cation exchange (CEX) chromatography resin.

[0196] Embodiment 77. The method according to any one of Embodiments 71 to 76, wherein a chromatography medium is packed into a chromatography column.

[0197] Embodiment 78. A method for improving the step yield of a frontal chromatography operation, To estimate the amount of protein in the loading solution, A method comprising determining the bed height of a frontal chromatography medium based on a target residence time, wherein the flow rate is a constant value in the range of 50 cm / hour to 250 cm / hour.

[0198] Embodiment 79. A method for improving the step yield of a frontal chromatography operation, To estimate the amount of protein in the loading solution, A method comprising determining the flow rate of a frontal chromatography operation based on a target residence time, wherein the flow rate is in the range of 50 cm / hour to 250 cm / hour, and the bed height of the frontal chromatography medium is constant in the range of 5 cm to 30 cm.

[0199] Embodiment 80. The method according to Embodiment 78 or Embodiment 79, wherein in some embodiments, the bed diameter of the frontal chromatography medium is kept constant.

[0200] Embodiment 81. The method according to any one of Embodiments 78 to 80, wherein the amount of protein in the loading solution is estimated based on monitoring performed in real time, near real time, and / or offline.

[0201] Embodiment 82. The method according to any one of Embodiments 78 to 80, wherein the amount of protein in the loading solution is estimated based on historical data.

[0202] Embodiment 83. The method according to any one of Embodiments 78 to 82, wherein the estimation is performed after one or more cycles.

[0203] Embodiment 84. The method according to any one of Embodiments 78 to 82, wherein the estimation is performed after one or more batches.

[0204] Embodiment 85. The method according to any one of Embodiments 78 to 84, wherein the bed diameter is in the range of 1 cm to 20 cm.

[0205] Embodiment 86. The method according to any one of Embodiments 78 to 85, wherein the bed diameter is 1 cm, 10 cm, or 20 cm.

[0206] Embodiment 87. The method according to any one of Embodiments 78 to 86, wherein the bed diameter is 1 cm.

[0207] Embodiment 88. The method according to any one of Embodiments 78 to 86, wherein the bed diameter is 10 cm.

[0208] Embodiment 89. The method according to any one of Embodiments 78 to 86, wherein the bed diameter is 20 cm.

[0209] Embodiment 90. The method according to any one of Embodiments 78 to 89, wherein the frontal chromatography medium is a cation exchange chromatography medium.

[0210] Embodiment 91. The method according to any one of Embodiments 78 to 90, wherein the frontal chromatography medium is a cation exchange chromatography resin.

[0211] Embodiment 92. The method according to any one of Embodiments 78 to 91, wherein the frontal chromatography medium is a cation exchange chromatography resin containing a tentacle ligand.

[0212] Embodiment 93. The method according to any one of Embodiments 78 to 92, wherein a frontal chromatography medium is packed into a chromatography column.

[0213] Embodiment 94. The method according to any one of Embodiments 1 to 93, wherein the protein is an antibody.

[0214] Embodiment 95. The method according to any one of Embodiments 1 to 94, wherein the protein is a monoclonal antibody.

[0215] Embodiment 96. The method according to any one of Embodiments 1 to 95, wherein the protein is an IgG2 antibody.

[0216] definition Where a range of values ​​is provided herein, unless the context explicitly indicates otherwise, each intervening value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated values ​​or intervening values ​​within that stated range, are understood to be included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in smaller ranges also included in this disclosure, subject to the limits specifically excluded in the stated range. If a stated range includes one or both limits, the range excluding either or both of those limits is also included in this disclosure.

[0217] As used herein, the terms “one (a)” and “one (an)” mean “one or more” unless otherwise indicated. Furthermore, “one or more” and “at least one” are used interchangeably herein. In addition, unless the context requires otherwise, singular terms include plural forms, and plural terms include singular forms.

[0218] Throughout this specification and the subsequent claims, unless the context requires otherwise, the word “comprise,” and variations such as “comprises” and “comprising,” mean to include the integer or step or group of integers or steps mentioned, but not to exclude any other integer or step or group of integers or steps. Where used herein, the term “comprising” may also be replaced by the terms “containing,” “including,” or, where used herein, “having.”

[0219] As used herein, the term "consisting of" excludes any element, step, or component not specified in the characteristics of the embodiment or the claimed element. As used herein, the term "essentially consisting of" does not exclude materials or steps that do not substantially affect the basic and novel features of the characteristics of the embodiment or the claimed element.

[0220] In each example herein, the terms “comprising,” “consisting essentially of,” and “consisting of,” and any variation thereof, may be replaced by one of the other two terms or any variation thereof.

[0221] As used herein, the term “affinity chromatography” (also known as “capture chromatography”) refers to a chromatographic operation that separates a biomolecule (e.g., recombinant protein) from a mixture based on the selective interaction between the biomolecule and another substance (i.e., a ligand). Affinity chromatography is commonly used in biomanufacturing processes to isolate and concentrate a desired recombinant protein from harvested cell cultures. In a typical affinity chromatography operation, a biomolecule in the mobile phase selectively binds to or otherwise interacts with the stationary phase, while the rest of the mobile phase passes through the chromatographic material. The biomolecule is then eluted from the stationary phase by changing the conditions to reduce the affinity between the ligand and the biomolecule. Non-limiting examples of affinity chromatography include protein A, protein G, protein A / G, and protein L materials. Furthermore, immobilized metal affinity chromatography (IMAC) may be used to capture proteins that have an affinity for metal ions, or that have been engineered to have an affinity for metal ions.

[0222] In some embodiments, protein A affinity chromatography can be used to capture the target protein. Protein A ligands are highly selective for a wide range of proteins containing antibody Fc regions, resulting in robust removal of process-related impurities and high target protein yield. Commercially available protein A materials include, but are not limited to, MABSELECT® SURE Protein A, Protein A Sepharose FAST FLOW®, MABSELECT® Prism A (Cytiva, Marborough, MA), PROSEP-A® (Merck Millipore, UK), TOYOPEARL® HC-650F Protein A (TosoHass Co., Philadelphia, PA), and AP Plus, Purolite, King of Prussia, PA.

[0223] As used herein, the term "bed height" refers to the height of the chromatography medium being used.

[0224] As used herein, the term “bed diameter” refers to the diameter of the chromatographic medium used. In some embodiments of the methods described herein, the bed diameter is greater than 1 cm, greater than 2 cm, greater than 3 cm, greater than 4 cm, greater than 5 cm, greater than 10 cm, greater than 15 cm, greater than 20 cm, greater than 25 cm, greater than 30 cm, greater than 35 cm, greater than 40 cm, or greater than 45 cm. In some embodiments, the bed diameter is 1 cm, 2 cm, 3 cm, 4 cm, 5 cm, 10 cm, 15 cm, 20 cm, 25 cm, 30 cm, 35 cm, 40 cm, or 45 cm. In some embodiments of the methods described herein, the bed diameter is greater than 80 cm, greater than 100 cm, or greater than 120 cm. In some embodiments, the bed diameter is 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 100 cm, 110 cm, 120 cm, 130 cm, 140 cm, 150 cm, 160 cm, 170 cm, 180 cm, 190 cm, or 200 cm. In some embodiments, the bed diameter is determined based on the amount of protein or contaminant being loaded.

[0225] As used herein, the term “bioreactor” means any vessel useful for growing mammalian cell cultures. As used herein, the term “fermenter” means any vessel useful for growing bacterial cell cultures, which typically include more rigorous agitators and increased gas flow compared to vessels used for growing mammalian cell cultures.

[0226] Non-limiting examples of bioreactors include agitated tank type, airlift type, fiber type, microfiber type, hollow fiber type, ceramic matrix type, fluid bed type, fixed bed type, and / or jet bed type bioreactors. In some embodiments, exemplary bioreactors may perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients and / or carbon sources, injecting suitable gases (e.g., oxygen), fermentation or inflow and outflow of cell media (e.g., supplying fresh cell media by perfusion and removing used cell media), separation of gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, stirring (e.g., agitation), and / or washing / sterilization. Unless otherwise indicated by context, bioreactors may be suitable for batch processes, semi-fed batch processes, fed batch processes, perfusion processes, and / or continuous fermentation processes. Any suitable bioreactor diameter may be used. Unless otherwise indicated by the context, in some embodiments, the bioreactor may have a volume of 100 mL to 50,000 L. Unless otherwise indicated by the context, the bioreactor may be of any size as long as it is useful for culturing cells; typically, the bioreactor is sized to suit the volume of the cell culture to be grown inside it. In non-limiting embodiments, and unless otherwise indicated by the context, the bioreactor may be at least 1 liter (L), or 2, 5, 10, 50, 100, 200, 250, 500, 1,000, 1,500, 2,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters, 20,000 L or more, or any volume in between. Internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen concentration, and temperature, may be controlled during the culture period. Those skilled in the art will be able to identify and select a bioreactor suitable for use in the manner disclosed herein, based on the relevant considerations.

[0227] As used herein, the term "buffer solution" refers to a solution that resists changes in pH due to the action of its acid-base conjugate components.

[0228] As used herein, the terms “cell culture” or “culture” refer to the proliferation and multiplication of cells outside of a multicellular organism or tissue. Suitable culture conditions for mammalian and bacterial cells are known in the art. (See, for example, Animal cell culture: A Practical Approach, D. Rickwood, ed., Oxford University Press, New York (1992)). Mammalian cells can be cultured in suspension or attached to solid culture media. In some embodiments, fluid bed bioreactors, hollow fiber bioreactors, roller bottles, shaking flasks and / or agitated tank bioreactors, with or without microcarriers, may be used for cell culture. In some embodiments, 500 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train). In some embodiments, 1000 L to 2000 L bioreactors are used for cell culture (e.g., as part of a seed train).

[0229] As used herein, the term “cell culture medium” (also referred to as “culture medium,” “culture medium,” “cell culture medium,” “tissue culture medium,” etc.) refers to any nutrient solution used to grow cells, such as bacterial or mammalian cells. A cell culture medium generally provides one or more of the following components: an energy source (e.g., in the form of carbohydrates such as glucose); one or more essential amino acids (e.g., all essential amino acids, the 20 basic amino acids plus cysteine); vitamins and / or other organic compounds usually required at low concentrations; lipids or free fatty acids; and trace elements such as inorganic compounds or naturally occurring elements usually required at very low concentrations, such as concentrations in the micromolar range. As used herein, a cell culture medium encompasses nutrient solutions that are typically and / or known to be used in any cell culture process, including but not limited to batch culture, extended batch culture, fed batch culture, intensive culture, and / or perfusion culture or continuous culture of cells.

[0230] As used herein, the term “cell density” refers to the number of cells in a given volume of culture medium. “Viable cell density” refers to the number of viable cells in a given volume of culture medium, as determined by a standard viability assay (e.g., trypan blue exclusion). As used herein, “packed cell volume” (PCV), also known as “percentage of packed cell volume” (%PCV), is the ratio of the volume occupied by cells to the total volume of a cell culture, and is expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). Packed cell volume is a function of cell density and cell diameter, and an increase in packed cell volume can occur due to an increase in cell density, cell diameter, or both. Packed cell volume is a measure of the solid content in a cell culture. Since host cells vary in size and cell cultures contain dead or dying cells and other cellular debris, the filled cell volume can more accurately describe the solid content within the cell culture.

[0231] As used herein, the terms “column” or “chromatographic column” refer to a device for separating components in a solution. A column consists of a stationary phase that adsorbs and separates components passing through a liquid mobile phase. Solid adsorbents, such as chromatography resins, are packed into glass or metal columns. Filtration-based adsorbents, such as membranes, and monoliths can function as stationary phases. The liquid phase contains the components to be separated (e.g., the product of interest, impurities, contaminants, etc.) in a buffer solution. As used herein, the term “medium volume” of a chromatography medium refers to the total volume of the chromatography medium used. For example, if the chromatography medium is packed into a column, the medium volume is the total volume of the chromatography medium in the column and is sometimes called the column volume. In all embodiments of this disclosure, the chromatography medium may be packed into a chromatography column, and references to “medium volume” or “MV” may be replaced with “column volume” or “CV.”

[0232] As used herein, the term “cycle” includes the steps of loading a loading solution containing the product of interest and at least one impurity onto a chromatographic medium, recovering the purified product of interest in the effluent after exiting the chromatographic column, and washing and equilibrating after recovery. A batch may have one or more cycles. As used herein, the term “batch” includes all chromatographic cycles necessary to process a certain volume of fluid containing the product of interest. The fluid may originate from elution pools or effluents from cell culture harvesting, affinity chromatography or polishing chromatography, virus inactivation, neutralization, deep filtration, viral filtration, ultrafiltration, dialysis filtration, or other upstream operations.

[0233] As used herein, the term “dynamic binding capacity” in relation to a chromatographic medium refers to the amount of product, such as polypeptides, to which the material will bind under actual flow conditions before significant intrusion of unbound products occurs.

[0234] As used herein, the term "loading" refers to a composition being loaded onto a chromatographic medium.

[0235] As used herein, the terms “loading,” “loading density,” “loading factor,” and “column loading” refer to the amount of the desired product (g / Lr) expressed in grams of the component per liter of resin. A flue flow or pool containing the desired product and at least one impurity may be loaded into the chromatography medium. Additional buffers may be added to the flue flow or pool to achieve a final loading volume of the desired concentration and / or formulation.

[0236] As used herein, the terms “expression vector” or “expression construct” refer to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid regulatory sequences necessary for the expression of an operably ligated coding sequence in a specific host cell, e.g., a mammalian host cell. Vectors may include viral vectors, non-episomal mammalian vectors, plasmids, and other nonviral vectors. Expression vectors may contain sequences that act on or control transcription, translation, and, where introns are present, act on RNA splicing of the coding region operably ligated thereto. “Operatably ligated” means that the components to which this term applies are related in such a way that they can perform their intrinsic functions. For example, a regulatory sequence in a vector “opertably ligated” to a protein coding sequence, such as a promoter, is positioned so that the normal activity of the regulatory sequence leads to the transcription of the protein coding sequence and the recombinant expression of the encoded protein.

[0237] As used herein, “fed-batch culture” refers to a form of suspension culture, specifically a method of culturing cells in which additional components are provided to the culture medium at some or more points after the start of the culture process. The supplied components typically include nutrient supplements for cells depleted during the culture process. In addition or alternatively, the additional components may include supplemental components (e.g., cell cycle inhibitors). In some embodiments, the fed-batch cell culture medium formulation may contain components essential for cell survival and proliferation and may be richer or more concentrated than the basal cell culture medium formulation typically used to initiate cell culture. The fed-batch culture may be stopped at some point, and the cells and / or components in the medium may be harvested and optionally purified.

[0238] As used herein, the “proliferative phase” of cell culture refers to the period of exponential cell proliferation (i.e., logarithmic phase) in which cells are generally dividing rapidly.

[0239] As used herein, the terms “harvested cell culture medium” or “harvested cell culture broth” refer to a solution treated by one or more operations to separate cells, cell debris, or other large particles from the protein of interest. Such operations include, but are not limited to, cooling, agglutination, acidification, centrifugation, neutralization, ultrasonic separation, and various forms of filtration (e.g., deep filtration, microfiltration, ultrafiltration, tangential flow filtration, and alternating tangential flow filtration). The harvested cell culture medium includes cell culture lysates and cell culture supernatants. The harvested cell culture medium may be further clarified to remove fine particulate matter and soluble aggregates by filtration through a membrane with a pore size of about 0.1 μm to about 0.5 μm, for example, a membrane with a pore size of about 0.22 μm.

[0240] As used herein, “host cell” refers to a cell that is transformed with nucleic acid, or can be transformed, to express the gene of interest. This term includes offspring of a parent cell, regardless of whether the offspring’s morphology or genetic structure is identical to that of the original parent cell, as long as the gene of interest is present. For example, a host cell containing nucleic acid encoding a recombinant protein, operably ligated to at least one expression regulatory sequence (e.g., a promoter or enhancer), is a “recombinant host cell.” When cultured under appropriate conditions, the host cell synthesizes a recombinant protein, which can then be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if the host cell does not secrete it).

[0241] As used herein, the terms “high molecular weight” or “HMW” species of the protein of interest refer to dimers, oligomers, and aggregates of the protein having a molecular weight greater than that of the intact, fully assembled form of the protein.

[0242] As used herein, the term “impurity” refers to components other than the protein of interest, along with their associated buffer components. Examples of impurities include, but are not limited to, host cell proteins, leached resin materials (e.g., leached protein A), nucleic acids, HMW species of proteins, LMW species of proteins, endotoxins, viral contaminants, cell culture medium components, and similar process and product-related impurities.

[0243] As used herein, the “low molecular weight” or “LMW” species of the recombinant protein of interest refers to a protein fragment, truncated form, or incomplete variant having a molecular weight smaller than that of the intact, fully assembled form of the protein. LMW species may include, but are not limited to, proteolytic fragments, truncated forms resulting from the cellular expression of mRNA splice variants, and single-component polypeptides in the case of multi-chain polypeptide proteins (e.g., light-chain or heavy-chain-only species if the recombinant protein is an antibody).

[0244] As used herein, “perfused” cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion or continuous culture methods and is sufficiently complete to support the cell culture during this process. In some embodiments, the perfused cell culture medium composition may be more concentrated or higher in concentration than the basic cell culture medium composition to accommodate the method used to remove used medium. In some embodiments, the perfused cell culture medium may be used in both the growth and production stages.

[0245] As used herein, the term "polishing chromatography" refers to a chromatographic operation performed after a capture chromatography or affinity chromatography operation to remove residual impurities and obtain a more highly purified composition and / or protein. Common impurities removed during the polishing step include, but are not limited to, product-related impurities (e.g., HMW and LMW species), host cell proteins, DNA, leached protein A, viral contaminants, and endotoxins. In addition, typical chromatographic techniques used for polishing include, but are not limited to, ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and multimodal (or mixed-mode) chromatography (MMC).

[0246] As used herein, “anion exchange chromatography” (AEX) refers to a form of ion exchange chromatography performed on a positively charged solid-phase medium (e.g., a resin or membrane) capable of exchanging free anions with anions in an aqueous solution passing over or through the solid phase. AEX chromatography is used, for example, for viral clearance and impurity removal. Commercially available anion exchange media include, but are not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15Q, Capto® Q, Q-SEPHAROSE FAST FLOW® (Cytiva), FRACTOGEL TMAE®, FRACTOGEL EMD DEAE® (EMD Merck), TOYOPEARL® Super Q®, and TOYOPEARL® NH2-750F (Tosoh Bioscience), POROS HQ®, and POROS XQ® (ThermoFisher).

[0247] As used herein, “cation exchange chromatography” (CEX) refers to a form of ion exchange chromatography performed on a negatively charged solid medium (e.g., a resin or membrane) capable of exchanging free cations with cations in an aqueous solution passing over or through the solid phase. The charge can be provided by attaching one or more charged ligands to the solid phase, for example, by covalent bonds. Alternatively or in addition, the charge may be an inherent property of the solid phase (e.g., silica with a total negative charge). CEX chromatography is typically used to remove high molecular weight (HMW) contaminants, process-related impurities, and / or viral contaminants. Commercially available cation exchange media include sulfopropyl (SP) immobilized on agarose (e.g., SPSEPHAROSE FAST FLOW (trademark), SP-SEPHAROSE FAST FLOW XL (trademark), or SP-SEPHAROSE HIGH PERFORMANCE (trademark), CAPTO S (trademark), CAPTO SP ImpRes (trademark), CAPTO S ImpAct (trademark) (Cytiva), FRACTOGEL-SO3 (trademark), FRACTOGEL-SE HICAP (trademark), and FRACTOPREP (trademark) (EMD Merck, Darmstadt, Germany), TOYOPEARL (registered trademark) XS, TOYOPEARL (registered trademark) HS (Tosoh Bioscience, King of Prussia, PA), UNOsphere (trademark) (BioRad, Hercules, CA), and S Ceramic Hyper (trademark) DF (Pall, Port). Examples include, but are not limited to, Washington, NY; POROS (Trademark) (ThermoFisher, Waltham, MA); ESHMUNO (Registered Trademark) CSP; and ESHMUNO (Registered Trademark) CP-FT (Millipore Sigma, Darmstadt, Germany).

[0248] As used herein, “hydrophobic interaction chromatography” (HIC) refers to chromatography performed on a solid-phase medium that utilizes the interaction between a hydrophobic ligand and a hydrophobic residue on the surface of a desired solute (e.g., a desired protein). Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sephrose® (Cytiva), Tosoh Hexyl (Tosoh Bioscience), and Capto® Phenyl (Cytiva).

[0249] As used herein, “mixed-mode or multimodal chromatography” (MMC) refers to chromatography that achieves separation by utilizing two or more forms of interaction between the stationary phase and the analyte. MMC differs from single-mode chromatography in that two or more interactions, such as electrostatic interactions, hydrogen bonding interactions, and / or hydrophobic interactions, significantly contribute to the retention of the solute. Commercially available multimodal chromatography media include, but are not limited to, Capto® Adhere, Capto® MMC Impress, Capto MMC, (Cytiva), PPA Hypercel, MEP Hypercell, HEA Hypercell (Pall Corporation, Port Washington, NY), Eshmuno HCX, (Merk Millipore), and TOYOPEARL® MX-Trp-650M (Tosoh Bioscience).

[0250] As used herein, “production” cell culture medium refers to a cell culture medium typically used in cell culture during the transition from exponential growth to a period of dominant protein production (i.e., the “transition” phase and / or “production” phase), which is sufficiently complete to maintain the desired cell density, viability, and / or biopotency during this phase. The production cell culture medium may be the same as or different from the cell culture medium used during the exponential growth phase of the cell culture.

[0251] As used herein, the “production stage” of cell culture refers to the period after logarithmic cell proliferation has ended and recombinant protein production has become dominant.

[0252] As used herein, the term “recombinant protein” refers to a heterologous protein produced when a host cell transfected with a protein-coding nucleic acid is cultured in a cell culture.

[0253] As used herein, the term “purified,” when used in relation to a composition, refers to a composition in which at least one impurity is present at a lower concentration compared to the composition in which it remained present before one or more unit operations. Furthermore, “purified” protein (e.g., purified antibody) refers to a protein that has been purified to a higher degree, meaning that it exists in a form of higher purity than when it was first synthesized and / or amplified under natural and / or laboratory conditions. Purity is a relative term and does not necessarily refer to absolute purity.

[0254] As used herein, the term “step yield” refers to the amount of product in the pool divided by the amount of loaded product. The amount of product in the pool is calculated as “(pool volume) × (pool concentration)” and is determined by UV measurement. The loading amount of product is calculated as “(loading amount) × (loading concentration)” and is determined by UV measurement. To determine both the pool concentration and the loading concentration, it can be assumed that the main molecular species contributing to the UV signature is the product of interest or its derivatives, for example, HMW impurities / aggregates have similar extinction coefficients, and other impurities either do not contribute to the UV signature or are present in amounts so small that their contribution to the UV signature is negligible.

[0255] As used herein, the term “titrate” refers to a solution of known concentration that is added to another solution during titration. “Acid titrate” refers to a titrate with a pH less than 7, while “basic titrate” refers to a titrate with a pH greater than 7.

[0256] As used herein, the term “unit operation” refers to a functional step performed as part of a process for purifying a protein of interest. A unit operation may be designed to achieve one or more purposes, such as a capture step, an acid precipitation step, a centrifugation step, or a chromatography step. A unit operation may also include holding or preserving steps between processing steps.

[0257] As used herein, the term “antibody” generally refers to a tetrameric immunoglobulin protein comprising two light chain polypeptides (each approximately 25 kDa) and two heavy chain polypeptides (each approximately 50–70 kDa). The term “light chain” or “immunoglobulin light chain” refers to a polypeptide comprising a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus. The immunoglobulin light chain constant domain (CL) may be a human kappa (κ) constant domain or a human lambda (λ) constant domain. The term “heavy chain” or “immunoglobulin heavy chain” refers to a polypeptide comprising a single immunoglobulin heavy chain variable region (VH), immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, immunoglobulin heavy chain constant domain 2 (CH2), immunoglobulin heavy chain constant domain 3 (CH3), and an optional immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus. Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG and IgA class antibodies are further divided into subclasses, namely IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2, respectively. The heavy chains of IgG, IgA, and IgD antibodies have three constant domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four constant domains (CH1, CH2, CH3, and CH4). The constant domains of immunoglobulin heavy chains may originate from any immunoglobulin isotype, including its subtypes. The antibody chains are linked to each other via polypeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light chain and the heavy chain) and between the hinge regions of the two antibody heavy chains.

[0258] The variable regions of immunoglobulin chains generally exhibit an identical overall structure, including a relatively conserved framework region (FR) linked by three hypervariable regions (more often called "complementarity-determining regions" or CDRs). The CDRs, derived from the two chains of each pair of heavy and light chains, are typically aligned by the framework region to form a structure that specifically binds to a particular epitope of a target protein. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions usually follow the following order of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Numbering systems have been devised to assign numbers to the amino acids occupying positions within each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or in Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. This system can be used to identify the CDR and FR of a given antibody. Other numbering systems for amino acids in immunoglobulin chains include IMGT (registered trademark) (the international ImMunoGeneTics information system; Lefranc et al., Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001).

[0259] When used in the context of the present invention, “binding fragment” or “antigen-binding fragment” as used interchangeably with “fragment” herein is a portion of an antibody that lacks at least some of the amino acids present in the full-length heavy chain and / or light chain, but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a heavy-chain-only antibody (e.g., camel heavy-chain antibody); VHH fragment, see Cortez-Retamozo et al., Cancer Research, Vol. 64: 2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and CDR fragments, and may originate from any mammalian source such as humans, mice, rats, rabbits, or camels. Antigen-binding fragments may compete with intact antibodies for binding to a target antigen, and the fragments may be produced by modifying intact antibodies (e.g., enzymatic or chemical cleavage) or newly synthesized using recombinant DNA technology or peptide synthesis. In some embodiments, the antigen-binding fragment comprises at least one CDR derived from the antibody that binds to the antigen, for example, a heavy chain CDR3 derived from the antibody that binds to the antigen. In other embodiments, the antigen-binding fragment comprises all three CDRs derived from the heavy chain of the antibody that binds to the antigen, or all three CDRs derived from the light chain of the antibody that binds to the antigen. In yet another embodiment, the antigen-binding fragment comprises all six CDRs derived from the antibody that binds to the antigen (three from the heavy chain and three from the light chain).

[0260] When an antibody is digested with papain, two identical antigen-binding fragments called "Fab" fragments (each possessing a single antigen-binding site) and the remainder, an "Fc" fragment (containing all but the first domain of the constant region of the immunoglobulin heavy chain), are produced. The Fab fragment contains the variable domains derived from the light chain and heavy chain, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the "Fab fragment" consists of one immunoglobulin light chain (variable region (VL) and constant region (CL)) and the CH1 domain and variable region (VH) of one immunoglobulin heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fd fragment" contains the VH domain and CH1 domain derived from the immunoglobulin heavy chain. The Fd fragment represents the heavy chain component of the Fab fragment.

[0261] An immunoglobulin “Fc fragment” or “Fc domain” generally comprises two constant domains, namely a CH2 domain and a CH3 domain, and optionally a CH4 domain. The Fc domain may be derived from IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc domain comprises CH2 and CH3 domains derived from human IgG1 or human IgG2 immunoglobulin. The Fc domain may retain effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), and phagocytosis. In other embodiments, the Fc domain may be modified to reduce or eliminate effector functions.

[0262] A "Fab fragment" is a Fab fragment that has one or more cysteine ​​residues derived from the hinge region of the antibody at the C-terminus of the CH1 domain.

[0263] The "F(ab')2 fragment" is a divalent fragment containing two Fab' fragments linked by disulfide bridges between heavy chains in the hinge region.

[0264] The "Fv" fragment is the smallest fragment containing the complete antigen recognition and binding sites derived from an antibody. This fragment consists of a dimer of one immunoglobulin heavy chain variable region (VH) and one immunoglobulin light chain variable region (VL) in a tight non-covalent state. In this configuration, the three CDRs of each variable region interact to define an antigen binding site on the surface of the VH-VL dimer. A single variable region of the light or heavy chain (or half of the Fv fragment containing only the three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but with lower affinity than the entire binding site containing both VH and VL.

[0265] A "single-chain variable fragment" or "scFv fragment" contains the VH and VL regions of an antibody, and these regions are present in a single polypeptide chain and optionally include a peptide linker between the VH and VL regions, thereby enabling the Fv to form the desired structure for antigen binding (see, for example, Bird et al., Science, Vol. 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA, Vol. 85:5879-5883, 1988).

[0266] "Nanobody" is the variable region of the heavy chain of a heavy chain antibody. Such variable domains are the smallest fully functional antigen-binding fragments of such heavy chain antibodies, with a molecular weight of only 15 kDa. See Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004. Functional heavy chain antibodies lacking light chains occur naturally in certain species of animals, such as sharks, rays, and the family Camelidae, such as camels, dromedaries, alpacas, and llamas. In these animals, the antigen-binding site is the VHH domain, which is a single domain. These antibodies form antigen-binding regions using only the variable region of the heavy chain, i.e., these functional antibodies are homodimers of only the heavy chain (also referred to as "heavy chain antibodies" or "HCAb"). Camelized VHHs have been reported to recombine with IgG2 and IgG3 constant regions lacking the CH1 domain and containing the hinge domain, CH2 domain, and CH3 domain. Camelized VHH domains have been found to bind antigens with high affinity (Desmyter et al., J. Biol. Chem., Vol. 276:26285-90, 2001) and have high stability in solution (Ewert et al., Biochemistry, Vol. 41:3628-36, 2002). Methods for generating antibodies with camelized heavy chains are described, for example, in U.S. Patent Application Publication Nos. 2005 / 0136049 and 2005 / 0037421. Alternative scaffolds can be created from human variable-like domains that fit more closely to the shark V-NAR scaffold and can provide long, penetrating loop structures in the framework. Human heavy chain antibodies, such as UniAb™ antibodies produced by UniRat™ transgenic rats, can be generated from transgenic animals expressing human immunoglobulin genes.

[0267] Chromatography medium The methods of the present disclosure utilize chromatographic media, including chromatographic media suitable for overload or frontal chromatography. Examples of chromatographic media that may be used in the methods of the present disclosure include, but are not limited to, ion exchange chromatography (IEX) media, including cation exchange chromatography (CEX) media and anion exchange chromatography (AEX) media, multimodal or mixed-mode chromatography (MMC) media, hydrophobic interaction chromatography (HIC) media, and hydroxyapatite (HA) media.

[0268] In some embodiments, the chromatography medium is a cation exchange chromatography medium. In some embodiments, the chromatography medium is a low ligand density cation exchange chromatography medium. In some embodiments, the chromatography medium is a cation exchange chromatography medium for frontal chromatography. In some embodiments, the chromatography medium is a cation exchange chromatography column.

[0269] In some embodiments, the chromatographic medium is a cation exchange chromatography medium with an ion density of less than 60 μeq / mL, less than 55 μeq / mL, less than 50 μeq / mL, less than 45 μeq / mL, or less than 40 μeq / mL. In some embodiments, the chromatographic medium is a cation exchange chromatography medium with an ion density of less than 55 μeq / mL, less than 50 μeq / mL, less than 45 μeq / mL, or less than 40 μeq / mL. In some embodiments, the chromatographic medium is a cation exchange chromatography medium with an ion density of less than 50 μeq / mL, less than 45 μeq / mL, or less than 40 μeq / mL. In some embodiments, the chromatographic medium is a cation exchange chromatography medium with an ion density of less than 45 μeq / mL or less than 40 μeq / mL. In some embodiments, the chromatographic medium is a cation exchange chromatography medium with an ion density of less than 40 μeq / mL. Ion density can be measured according to the method described in Stone, Matthew T., Kristen A. Cotoni, and Jayson L. Stoner, “Cation exchange frontal chromatography for the removal of monoclonal antibody aggregates,” Journal of Chromatography A 1599(2019):152-160.

[0270] In some embodiments, the chromatography medium is an anion exchange chromatography medium. In some embodiments, the chromatography medium is an anion exchange chromatography medium for frontal chromatography. In some embodiments, the chromatography medium is an anion exchange chromatography column.

[0271] In some embodiments, the chromatography medium is an MMC medium. In some embodiments, the chromatography medium is an MMC medium for frontal chromatography. In some embodiments, the chromatography medium is an MMC column.

[0272] In some embodiments, the chromatography medium is an HIC medium. In some embodiments, the chromatography medium is an HIC medium for frontal chromatography. In some embodiments, the chromatography medium is an HIC column.

[0273] In some embodiments, the chromatography medium is packed into the chromatography column.

[0274] In some embodiments, the chromatography medium is a membrane, a monolith, or a resin. In some embodiments, the chromatography medium is a membrane. In some embodiments, the chromatography medium is a monolith. In some embodiments, the chromatography medium is a resin.

[0275] In some embodiments, the bed diameter of the chromatography medium is 50 cm, 60 cm, 70 cm, 80 cm, 90 cm, 100 cm, 110 cm, 120 cm, 130 cm, 140 cm, 150 cm, 160 cm, 170 cm, 180 cm, 190 cm, or 200 cm.

[0276] In some embodiments, the bed diameter of the chromatography medium is determined based on the amount of products and / or contaminants being loaded.

[0277] The bed height of the chromatography medium can be varied to adjust the medium volume and achieve a target loading density based on the amount of influent product, which can be measured directly or estimated based on past titers. Changing the bed height may be a more feasible means of adjusting the medium volume and the resulting loading density than changing the bed diameter. This is because, unlike changing the column diameter, changing the bed height does not require changes to the equipment or the plant layout and / or footprint, as manufacturing sites typically have limited options for changing hardware specifications, equipment availability, and plant layout.

[0278] In some embodiments, the chromatography medium has a bed height of at least 5 cm. In some embodiments, the chromatography medium has a bed height in the range of 5 cm to 30 cm. In some embodiments, the chromatography medium has a bed height in the range of 5 cm to 15 cm.

[0279] The chromatography medium has a bed height of 5cm, 6cm, 7cm, 8cm, 9cm, 10cm, 11cm, 12cm, 13cm, 14cm, 15cm, 16cm, 17cm, 18cm, 19cm, 20cm, 21cm, 22cm, 23cm, 24cm, 25cm, 26cm, 27cm, 28cm, 29cm, or 30cm. In some embodiments, the chromatography medium has a bed height of 10cm, 11cm, 12cm, 13cm, 14cm, or 15cm.

[0280] In some embodiments, the bed height of the chromatography medium is determined based on the amount of products and / or contaminants being loaded. In some embodiments, the bed height of the chromatography medium is determined based on the target residence time. In some embodiments, the bed height of the chromatography medium is determined based on the target residence time, and the flow rate is a constant value in the range of 50 cm / hour to 250 cm / hour (e.g., 50 cm / hour, 75 cm / hour, 100 cm / hour, 125 cm / hour, 150 cm / hour, 175 cm / hour, 200 cm / hour, 225 cm / hour, 250 cm / hour, etc.).

[0281] In some embodiments, the chromatography media contains at least 1 mL, at least 2 mL, at least 3 mL, at least 4 mL, at least 5 mL, at least 6 mL, at least 7 mL, at least 8 mL, at least 9 mL, at least 10 mL, at least 15 mL, at least 20 mL, at least 25 mL, at least 30 mL, at least 40 mL, at least 50 mL, at least 75 mL, at least 100 mL, at least 200 mL, at least 300 mL, at least 400 mL, at least 500 mL, and at least 600 mL. A column having a volume of L, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, at least 2 L, at least 3 L, at least 4 L, at least 5 L, at least 6 L, at least 7 L, at least 8 L, at least 9 L, at least 10 L, at least 25 L, at least 50 L, at least 100 L, at least 200 L, at least 300 L, at least 400 L, at least 500 L, at least 600 L, at least 700 L, at least 800 L, at least 900 L, or at least 1000 L.

[0282] equilibration solution The chromatographic medium can be equilibrated with an equilibration solution before loading. The selection of a suitable equilibration solution and / or concentration of the equilibration solution is within the capabilities of those skilled in the art. In some embodiments, the equilibration solution contains 50 mM acetate and has a pH of 5.0.

[0283] In some embodiments, the equilibration solution contains acetate. In some embodiments, the equilibration solution contains acetate at concentrations in the range of 10 mM to 100 mM, 20 mM to 100 mM, 30 mM to 100 mM, 40 mM to 100 mM, 10 mM to 90 mM, 10 mM to 80 mM, 10 mM to 70 mM, 10 mM to 60 mM, 20 mM to 60 mM, or 30 mM to 60 mM.

[0284] In some embodiments, the equilibration solution contains acetate at a concentration of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0285] In some embodiments, the pH of the equilibration solution ranges from 4.0 to 8.0, 5.0 to 8.0, 6.0 to 8.0, 7.0 to 8.0, 4.0 to 7.0, 4.0 to 6.0, 4.0 to 5.0, or 5.0 to 6.0. In some embodiments, the pH of the equilibration solution is 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the pH of the equilibration solution is 5.0.

[0286] In some embodiments, before loading the composition, an equilibration solution in an amount 3 to 10 times, 3 to 9 times, 3 to 8 times, 3 to 7 times, 3 to 6 times, 3 to 5 times, 3 to 4 times, 4 to 10 times, 4 to 9 times, 4 to 8 times, 4 to 7 times, 4 to 6 times, 4 to 5 times, 5 to 10 times, 6 to 10 times, 6 to 9 times, 6 to 8 times, 6 to 7 times, 7 to 10 times, 7 to 9 times, 7 to 8 times, 8 to 10 times, 8 to 9 times, or 9 to 10 times the volume of the medium (e.g., column volume) is passed through the chromatography medium. In some embodiments, before loading the composition, an equilibration solution in an amount 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times the volume of the medium (e.g., column volume) is passed through the chromatography medium.

[0287] Loading solution Some embodiments of the present disclosure involve loading a composition, such as a loading solution containing proteins and one or more impurities, onto a chromatographic medium. The loading solution may originate from an efflux and / or product pool derived from a previous upstream unit operation such as cell culture harvesting, capture chromatography, affinity chromatography, polishing chromatography, virus inactivation, neutralization, deep filtration, viral filtration, UF / DF, or other operations. Prior to loading, the loading feed may be diluted or conditioned to achieve a target pH, conductivity, buffer composition, etc., of the loading solution. In some embodiments, the loading solution originates from an efflux or product pool from a high-volume chromatographic medium capable of high-column loading. Non-exclusive examples of high-volume chromatography media include MabSelect® PrismA®, Capto® Q (Cytiva, Marlborough, MA), PRAESTO® Jetted resin (Purolite, King of Prussia, PA), Porous® XQ (Waltham, MA), Eshmuno® Q (EMD Millipore, Burlington, MA), TOYOPEARL® GigaCap, and TOYOPEARL® NH2-750F (Tosoh Bioscience, Tokyo, Japan).

[0288] The concentration of the target product in the loading solution can be determined by measuring the UV absorbance at 280 nm and / or A300 to determine the loading volume for each cycle of the frontal loading chromatography steps. In some embodiments, the concentration of the target product is suitable for meeting the production schedule and / or timeline. In some embodiments, the concentration of the target product in the loading solution is 5 g / L or higher.

[0289] The selection of an appropriate loading solution and / or loading solution concentration is within the capabilities of those skilled in the art to which this disclosure relates. Such loading solutions may include, but are not limited to, acetates, citrates, phosphates, glycine, L-arginine, L-histidine, and 2-(N-morpholino)ethanesulfonic acid (MES). In some embodiments, the loading solution and / or loading solution concentration is the same as the outflow and / or pool containing the target product from a previous upstream operation.

[0290] In some embodiments, the loading solution contains acetate. In some embodiments, the loading solution contains acetate at a concentration in the range of 25 mM to 200 mM. In some embodiments, the loading solution contains acetate at a concentration in the range of 50 mM to 100 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 25 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 30 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 35 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 40 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 45 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 50 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 55 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 60 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 65 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 70 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 75 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 80 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 85 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 90 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 95 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 100 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 125 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 150 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 175 mM. In some embodiments, the loading solution contains acetate at a concentration of at least 200 mM.

[0291] In some embodiments, the loading solution contains acetate at concentrations of 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0292] In some embodiments, the loading solution contains acetate at a concentration of 50 mM. In some embodiments, the loading solution contains acetate at a concentration of 51 mM. In some embodiments, the loading solution contains acetate at a concentration of 52 mM. In some embodiments, the loading solution contains acetate at a concentration of 53 mM. In some embodiments, the loading solution contains acetate at a concentration of 54 mM. In some embodiments, the loading solution contains acetate at a concentration of 55 mM. In some embodiments, the loading solution contains acetate at a concentration of 56 mM. In some embodiments, the loading solution contains acetate at a concentration of 57 mM. In some embodiments, the loading solution contains acetate at a concentration of 58 mM. In some embodiments, the loading solution contains acetate at a concentration of 59 mM. In some embodiments, the loading solution contains acetate at a concentration of 60 mM. In some embodiments, the loading solution contains acetate at a concentration of 65 mM. In some embodiments, the loading solution contains acetate at a concentration of 70 mM. In some embodiments, the loading solution contains acetate at a concentration of 75 mM. In some embodiments, the loading solution contains acetate at a concentration of 80 mM. In some embodiments, the loading solution contains acetate at a concentration of 85 mM. In some embodiments, the loading solution contains acetate at a concentration of 90 mM. In some embodiments, the loading solution contains acetate at a concentration of 95 mM. In some embodiments, the loading solution contains acetate at a concentration of 100 mM.

[0293] The pH of the loading solution can affect binding and impurity clearance by regulating the affinity of the loading components to the chromatographic medium. In some embodiments, the pH of the loading solution is in the range of 3.6 to 6.0. In some embodiments, the pH is in the range of 4.0 to 5.6. In some embodiments, the pH is in the range of 4.5 to 5.6. In some embodiments, the pH is in the range of 5.0 to 5.6. In some embodiments, the pH is in the range of 4.5 to 5.5. In some embodiments, the pH is at least 4.0. In some embodiments, the pH is at least 4.1. In some embodiments, the pH is at least 4.2. In some embodiments, the pH is at least 4.3. In some embodiments, the pH is at least 4.4. In some embodiments, the pH is at least 4.5. In some embodiments, the pH is at least 4.6. In some embodiments, the pH is at least 4.7. In some embodiments, the pH is at least 4.8. In some embodiments, the pH is at least 4.9. In some embodiments, the pH is at least 5.0. In some embodiments, the pH is at least 5.1. In some embodiments, the pH is at least 5.2. In some embodiments, the pH is at least 5.3. In some embodiments, the pH is at least 5.4. In some embodiments, the pH is about 5.5. In some embodiments, the pH is about 5.6. In some embodiments, the pH is 3.6, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In some embodiments, the pH of the loading solution is at least 6.0. In some embodiments, formic acid is used to maintain the pH of the loading solution.

[0294] The conductivity of the loading solution can affect binding and impurity clearance. Loading feeds using low-conductivity loading solutions may promote stronger protein binding during loading. Lower conductivity reduces the number of conditioning cycles and allows for higher protein concentrations, both of which improve facility suitability (i.e., enabling a smaller overall pool volume that facilitates a more enhanced process). In some embodiments, the conductivity of the loading solution is 10 mS / cm or less, for example, 9 mS / cm or less, 8 mS / cm or less, 7 mS / cm or less, 6 mS / cm or less, or 5 mS / cm or less.

[0295] In some embodiments, the conductivity is in the range of 4 mS / cm to 10 mS / cm, for example, in the range of 4 mS / cm to 5 mS / cm. In some embodiments, the conductivity is 4.1 mS / cm, 4.2 mS / cm, 4.3 mS / cm, 4.4 mS / cm, 4.5 mS / cm, 4.6 mS / cm, 4.7 mS / cm, 4.8 mS / cm, 4.9 mS / cm, 5.0 mS / cm, 5.1 mS / cm, 5.2 mS / cm, 5.3 mS / cm, 5.4 mS / cm, 5.5 mS / cm, 5.6 mS / cm, 5.7 mS / cm, 5.8 mS / cm, 5.9 mS / cm, 6.0 mS / cm, 6.1 mS / cm, 6.2 mS / cm, 6.3 mS / cm, 6.4 mS / cm, 6.5 mS / cm, 6.6 mS / cm, 6.7 mS / cm, 6.8 mS / cm, 6.9 mS / cm, 7.0 mS / cm, 7.1mS / cm, 7.2mS / cm, 7.3mS / cm, 7.4mS / cm, 7.5mS / cm, 7.6mS / cm, 7.7mS / cm, 7 .8mS / cm, 7.9mS / cm, 8.0mS / cm, 8.1mS / cm, 8.2mS / cm, 8.3mS / cm, 8.4mS / cm, 8.5mS / cm, The values ​​are 8.6 mS / cm, 8.7 mS / cm, 8.8 mS / cm, 8.9 mS / cm, 9.0 mS / cm, 9.1 mS / cm, 9.2 mS / cm, 9.3 mS / cm, 9.4 mS / cm, 9.5 mS / cm, 9.6 mS / cm, 9.7 mS / cm, 9.8 mS / cm, 9.9 mS / cm, or 10.0 mS / cm.

[0296] In some embodiments, the conductivity is 4.0 mS / cm. In some embodiments, the conductivity is 4.1 mS / cm. In some embodiments, the conductivity is 4.2 mS / cm. In some embodiments, the conductivity is 4.3 mS / cm. In some embodiments, the conductivity is 4.4 mS / cm. In some embodiments, the conductivity is 4.5 mS / cm. In some embodiments, the conductivity is 4.6 mS / cm. In some embodiments, the conductivity is 4.7 mS / cm. In some embodiments, the conductivity is 4.8 mS / cm. In some embodiments, the conductivity is 4.9 mS / cm. In some embodiments, the conductivity is 5.0 mS / cm.

[0297] In some embodiments, the conductivity of the loading solution can be adjusted by using a low-buffering buffer. In some embodiments, the loading solution has low buffering capacity. In some embodiments, the conductivity of the loading solution may be established by the conditions under which the loading feed is derived. In some embodiments, an acid can be used that can establish and maintain low buffering capacity for the loading feed. In some embodiments, the acid can establish and maintain low buffering capacity for the loading feed resulting from a low-pH virus inactivation operation. In some embodiments, the acid is formic acid. The combination of a low-buffering buffer and formic acid can also be used to minimize the amount of base titrant (e.g., 2M Tris base or similar base titrant) required to raise the pH to target conditions suitable for use in a chromatographic medium (e.g., a cation exchange chromatography medium), thereby minimizing the increase in the conductivity of the loading feed. The decrease in the conductivity of the loading feed can also be achieved by diluting the loading feed with water or another suitable buffer.

[0298] Loading density A characteristic of frontal chromatography is that the target product binds significantly initially (i.e., at a low loading density), and then, at a higher loading density, the bound target product is replaced by more affinity impurities. The replaced target product flows into the effluent, the target product is concentrated, and recovered as a product pool. Therefore, the step yield of a frontal loading chromatography operation can vary non-linearly with respect to loading.

[0299] If the loading density is low, a significant portion of the products loaded into the resin may remain bound and cannot be recovered, resulting in a low yield. Conversely, if the loading density is high, the resin becomes more saturated with impurities, increasing the proportion of products that leach out and leading to a more linear increase in yield.

[0300] Before commencing the chromatographic operation, the product concentration of the loading solution can be determined by measuring the absorbance at 280 nm or 300 nm. The product concentration of the loading solution may be used to determine the loading volume for each cycle of the frontal chromatography step.

[0301] Some embodiments of this disclosure involve loading a composition, such as a loading solution containing a protein and one or more impurities, onto a chromatographic medium. In some embodiments, the loading density is at least 200 g / Lr. In some embodiments, the loading density is 1000 g / Lr or more. In some embodiments, the loading density is in the range of 1000 g / Lr to 1500 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 1500 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 1100 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 1000 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 950 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 900 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 850 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 800 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 750 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 700 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 650 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 600 g / Lr. In some embodiments, the loading density is in the range of 500 g / Lr to 550 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 1,100 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 1,000 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 950 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 900 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 850 g / Lr.In some embodiments, the loading density is in the range of 700 g / Lr to 800 g / Lr. In some embodiments, the loading density is in the range of 700 g / Lr to 750 g / Lr.

[0302] In some embodiments, the loading density is at least 200, at least 300, at least 400, at least 500, at least 525, at least 550, at least 575, at least 600, at least 625, at least 650, at least 675, at least 700, at least 725, at least 750, at least 775, at least 800, at least 825, at least 850, at least 875, at least 900, at least 925, at least 950, at least 975, at least 1,000, at least 1,100, at least 1,200, at least 1,300, at least 1,400, at least 1,500, at least 2,000, or at least 3,000 g / Lr. In some embodiments, the loading density is at least 500 g / Lr. In some embodiments, the loading density is at least 525 g / Lr. In some embodiments, the loading density is at least 550 g / Lr. In some embodiments, the loading density is at least 575 g / Lr. In some embodiments, the loading density is at least 600 g / Lr. In some embodiments, the loading density is at least 625 g / Lr. In some embodiments, the loading density is at least 650 g / Lr. In some embodiments, the loading density is at least 675 g / Lr. In some embodiments, the loading density is at least 700 g / Lr. In some embodiments, the loading density is at least 725 g / Lr. In some embodiments, the loading density is at least 750 g / Lr. In some embodiments, the loading density is at least 775 g / Lr. In some embodiments, the loading density is at least 800 g / Lr. In some embodiments, the loading density is at least 825 g / Lr. In some embodiments, the loading density is at least 850 g / Lr. In some embodiments, the loading density is at least 875 g / Lr. In some embodiments, the loading density is at least 900 g / Lr.In some embodiments, the loading density is at least 925 g / Lr. In some embodiments, the loading density is at least 950 g / Lr. In some embodiments, the loading density is at least 975 g / Lr. In some embodiments, the loading density is at least 1,000 g / Lr. In some embodiments, the loading density is at least 1,100 g / Lr. In some embodiments, the loading density is at least 1,200 g / Lr, and in some embodiments, the loading density is at least 1,300 g / Lr. In some embodiments, the loading density is at least 1,400 g / Lr. In some embodiments, the loading density is at least 1,500 g / Lr. In some embodiments, the loading density is at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,100, at least 1,500, at least 2,000, or at least 3,000 g / Lr.

[0303] In some embodiments, the loading density is 200, 300, 400, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 2,000, or 3,000 g / Lr.

[0304] In some embodiments, the loading density is sufficient to achieve a step yield in the range of 60% to 100%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 70%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 80%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 90%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 92.5%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 95%. In some embodiments, the loading density is sufficient to achieve a step yield of at least 97.5%.

[0305] In some embodiments, the loading density is sufficient to achieve a concentration of high molecular weight species in the product pool that is at least 0.5% lower compared to the loading feed. In some embodiments, the concentration of HMW molecular species in the product pool is in the range of 1% to 10% lower compared to the loading feed. In some embodiments, the loading density is sufficient to achieve a concentration of high molecular weight species in the product pool that is at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 7.5%, at least 8%, at least 8.5%, at least 9%, and at least 9.5% lower compared to the loading feed.

[0306] In some embodiments, loading is sufficient to achieve concentrations of high molecular weight species of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

[0307] In some embodiments, the concentration of HMW molecular species is 1% lower in the product pool compared to the loading feed. In some embodiments, the concentration of HMW molecular species is 2% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 3% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 4% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 5% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 6% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 7% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 8% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 9% lower in the product pool than in the loading feed. In some embodiments, the concentration of HMW molecular species is 10% lower in the product pool than in the loading feed.

[0308] In some embodiments, the concentration of HMW molecular species is 1% to 10% lower in the product pool compared to the loading feed.

[0309] In some embodiments, the loading density is sufficient to achieve a host cell protein impurity concentration such that the product pool is at least 1 / 1.5 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration in the product pool that is in the range of 1 / 2 to 1 / 10 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration such that the product pool is at least 1 / 2 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration such that the product pool is at least 1 / 3 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration such that the product pool is at least 1 / 4 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration such that the product pool is at least 1 / 5 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration such that the product pool is at least 1 / 6 of the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration in the product pool that is at least one-seventh of that in the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration in the product pool that is at least one-eighth of that in the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration in the product pool that is at least one-ninth of that in the loading feed. In some embodiments, the loading density is sufficient to achieve an HCP impurity concentration in the product pool that is one-tenth of that in the loading feed.

[0310] Recovery of the target product Effluent containing the desired product from a frontal chromatography operation can be reused for loading into another unit operation or recovered as a product pool. Effluent containing the desired product can also be recovered as a fixed quantity. Recovery criteria can be any desired endpoint, such as product and / or impurity concentration, volume, time, process and / or production parameters.

[0311] In some embodiments, the spillage containing the target product can be recovered over time as individual fractions. These fractions containing the target product can be stored as separate product pools or combined into a single product pool.

[0312] In some embodiments, the spill is monitored by UV. The selection of an appropriate UV absorbance criterion is within the capabilities of those skilled in the art. In some embodiments, the spill is discarded until the UV absorbance reaches a certain absorbance and product recovery is initiated. In some embodiments, the recovery initiation criterion is based on absorbance. In some embodiments, the spill is monitored at A280 (i.e., absorbance at 280 nm) and product recovery is initiated at a certain absorbance value. In some embodiments, product recovery is initiated at a fixed absorbance value in the range of 0.4 AU / cm to 0.6 AU / cm at A280. In some embodiments, product recovery is initiated at a fixed absorbance value in the range of 0.4 AU / cm to 0.5 AU / cm at A280. In some embodiments, product recovery is initiated at a fixed absorbance value in the range of 0.5 AU / cm to 0.6 AU / cm at A280. In some embodiments, product recovery is initiated at a fixed absorbance value of at least 0.5 AU / cm at A280. In some embodiments, product recovery is initiated at a fixed absorbance value of 0.5 AU / cm at A280. In some embodiments, product recovery is initiated at a fixed absorbance value of 0.6 AU / cm at A280.

[0313] The quality of the products in each product pool can be optionally determined for each run using methods and apparatus known in the art.

[0314] As loading progresses, the chromatographic medium becomes saturated with highly affinity impurities, and the effluent becomes concentrated with the target product. Beyond this saturation point, additional impurities loaded onto the chromatographic medium are expected to flow into the effluent along with the target product. The saturation point of the critical impurity designed to bind during the frontal loading chromatography steps (e.g., HMW product-related impurities) typically represents a practical upper limit for column loading. In some embodiments, effluent recovery is stopped just before or at this upper limit.

[0315] In some embodiments, the recovery of spilled material can be monitored by UV absorbance. When the UV absorbance reaches a certain percentage of the peak value at a given UV absorbance, the recovery of spilled material can be stopped.

[0316] In some embodiments, the UV absorbance is A280 (280 nm). In some embodiments, the recovery stop criterion is 40-60% of the peak maximum value at A280. In some embodiments, the recovery stop criterion is 40-50% of the peak maximum value at A280. In some embodiments, the recovery stop criterion is 50-60% of the peak maximum value at A280. In some embodiments, the recovery stop criterion is 40% of the peak maximum value at A280. In some embodiments, the recovery stop criterion is 50% of the peak maximum value at A280. In some embodiments, the recovery stop criterion is 60% of the peak maximum value at A280.

[0317] In some embodiments, the UV absorbance is A300. In some embodiments, the recovery stop criterion is 20-50% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is 40-50% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is at least 20% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is at least 30% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is at least 40% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is at least 45% of the peak maximum value at A300. In some embodiments, the recovery stop criterion is at least 50% of the peak maximum value at A300.

[0318] The criteria for stopping the recovery process may be based on a certain optical density (OD). In some embodiments, the certain OD is in the range of 0.2 to 3.0.

[0319] Rinse after loading Once loading is complete, the chromatography medium may be subjected to an optional post-loading rinse to recover any remaining unbound products. The effluent from the post-loading rinse, including the products, may be recovered and stored as a separate product pool, or it may be recovered and added to the loading product pool. The rinse effluent may be recovered as individual fractions over time or as a fixed volume.

[0320] The selection of an appropriate post-loading rinse solution and / or concentration is within the capabilities of those skilled in the art. In some embodiments, the post-loading rinse solution is the same as the unmodified rinse solution. In some embodiments, the post-loading rinse contains acetate. In some embodiments, the post-loading rinse contains acetate in a concentration ranging from 50 nM to 100 mM. In some embodiments, the post-loading rinse solution contains salt. In some embodiments, the post-loading rinse solution contains sodium chloride. In some embodiments, the post-loading rinse solution contains sodium chloride in a concentration of 20 mM or less.

[0321] In some embodiments, the conductivity of the post-loading rinse solution is greater than or equal to the conductivity of the loading solution. In some embodiments, the conductivity of the post-loading rinse solution is 5.0 mS / cm or greater. In some embodiments, the conductivity of the post-loading rinse solution is in the range of 5.0 mS / cm to 10.0 mS / cm, for example, in the range of 5.0 mS / cm to 6.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.0 mS / cm.

[0322] In some embodiments, the conductivity of the post-loading rinse solution is 5.0 mS / cm, 5.1 mS / cm, 5.2 mS / cm, 5.3 mS / cm, 5.4 mS / cm, 5.5 mS / cm, 5.6 mS / cm, 5.7 mS / cm, 5.8 mS / cm, 5.9 mS / cm, 6.0 mS / cm, 6.5 mS / cm, 7.0 mS / cm, 7.5 mS / cm, 8.0 mS / cm, 8.5 mS / cm, 9.0 mS / cm, 9.5 mS / cm, or 10.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.1 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.2 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.3 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.4 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.5 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.6 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.7 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.8 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 5.9 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 6.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 6.5 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 7.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 7.5 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 8.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 8.5 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 9.0 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 9.5 mS / cm. In some embodiments, the conductivity of the post-loading rinse solution is 10.0 mS / cm.

[0323] In some embodiments, the pH of the post-loading rinse solution is equal to or greater than the pH of the loading solution. In some embodiments, the pH of the post-loading rinse solution is 5.0 or higher. In some embodiments, the pH is in the range of 5.0 to 5.6. In some embodiments, the pH is at least 5.0. In some embodiments, the pH is at least 5.1. In some embodiments, the pH is at least 5.2. In some embodiments, the pH is at least 5.3. In some embodiments, the pH is at least 5.4. In some embodiments, the pH is about 5.5. In some embodiments, the pH is at least 5.6.

[0324] In some embodiments, the pH is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or 5.6. In some embodiments, the pH is about 5.0. In some embodiments, the pH is about 5.1. In some embodiments, the pH is about 5.2. In some embodiments, the pH is about 5.3. In some embodiments, the pH is about 5.4. In some embodiments, the pH is about 5.5. In some embodiments, the pH is about 5.6.

[0325] In some embodiments, the amount recovered from the post-loading rinse is less than or equal to 1.0 times the volume (MV) of the rinse medium to limit the introduction of impurities into the final product pool. In some embodiments, a rinse solution of 0.1 MV to 1.0 MV is applied to the column. In some embodiments, a rinse solution of 0.2 MV is applied to the column. In some embodiments, a rinse solution of 0.3 MV is applied to the column. In some embodiments, a rinse solution of 0.4 MV is applied to the column. In some embodiments, a rinse solution of 0.5 MV is applied to the column. In some embodiments, a rinse solution of 0.6 MV is applied to the column. In some embodiments, a rinse solution of 0.7 MV is applied to the column. In some embodiments, a rinse solution of 0.8 MV is applied to the column. In some embodiments, a rinse solution of 0.9 MV is applied to the column. In some embodiments, a rinse solution of 1.0 MV is applied to the column.

[0326] In some embodiments, a stop criterion for recovering the product-containing effluent from the post-loading rinse is achieved when the UV absorbance reaches a certain percentage of the maximum value observed during product loading. In some embodiments, the recovery stop criterion is in the range of 75% to 85% of the peak maximum in A300. In some embodiments, the recovery stop criterion is in the range of 80% to 85% of the peak maximum in A300. In some embodiments, the recovery stop criterion is at least 75% of the peak maximum in A300. In some embodiments, the recovery stop criterion is at least 80% of the peak maximum in A300. In some embodiments, the recovery stop criterion is at least 85% of the peak maximum in A300. In some embodiments, the recovery stop criterion is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, or 85% of the peak maximum in A300.

[0327] In some embodiments, the recall stop criterion is in the range of 40% to 60% of the peak maximum in A300. In some embodiments, the recall stop criterion is in the range of 50% to 60% of the peak maximum in A300. In some embodiments, the recall stop criterion is at least 40% of the peak maximum in A300. In some embodiments, the recall stop criterion is at least 45% of the peak maximum in A300. In some embodiments, the recall stop criterion is at least 50% of the peak maximum in A300. In some embodiments, the recall stop criterion is at least 55% of the peak maximum in A300. In some embodiments, the recall stop criterion is at least 60% of the peak maximum in A300. In some embodiments, the recovery stop criterion is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, or 60% of the peak value at A300.

[0328] Alternatively, the criteria for halting the recall may be based on a certain optical density (OD), such as an OD in the range of 0.2 to 3.0.

[0329] Washing of chromatography media Washing extends the functional life of the chromatography medium, allowing it to be reused for multiple cycles. Washing of chromatography medium may consist of one or more non-denaturing washing steps and / or one or more denaturing regenerating washing steps. The non-denaturing washing steps remove bound impurities from the chromatography medium, while the denaturing washing steps further remove bound impurities and disinfect the chromatography medium for reuse or storage.

[0330] The linear salt concentration gradient washing method described herein is useful for non-denaturing washing of chromatographic media that are highly saturated with impurities related to the product (e.g., chromatographic media operated in frontal chromatography mode at a high loading density; chromatographic media operated in a mode in which a nonlinear yield vs. loading density is observed). In some embodiments, the chromatographic media is highly saturated with impurities at the end of the chromatographic operation cycle.

[0331] In some embodiments, the chromatography medium is exposed to a high loading density during the chromatography operation. In some embodiments, a linear salt concentration gradient allows for slower desorption and elution of bound impurities compared to a washing step with a uniform concentration. Washing with a uniform concentration causes rapid desorption of bound impurities, potentially leading to column pressure exceeding the hardware's capacity. In contrast, linear salt concentration gradient washing can stabilize the operating pressure and reduce fouling of the chromatographic medium. Washing with a linear salt concentration gradient as described herein can be used alone as a non-denaturing washing step or in combination with a denaturing washing step with a uniform concentration to further remove bound impurities and / or contaminants from the chromatographic medium for reuse.

[0332] In some embodiments, bound impurities include process-related impurities, which typically originate within the manufacturing process and include cell substrates such as host cell proteins, nucleic acids (e.g., chromosomal or extrachromosomal DNA, t-RNA, rRNA, or mRNA), lipids (e.g., cell wall materials), cell culture components (e.g., culture medium components, serum, inducers, antibiotics, surfactants, defoaming waste, etc.), chromatography media used in other purification operations (e.g., protein A ligands), solvents, buffer components, indefins such as endotoxins and viruses, or combinations thereof, as well as extracts and / or elutes (e.g., β-glucan in depth filters). In some embodiments, bound impurities may also consist of product-related impurities, such as variants of the target product that do not share common properties with the target product in terms of activity, efficacy, and / or safety, and in such cases, their presence in the purified active pharmaceutical ingredient or final formulation may adversely affect the quality, safety, and / or efficacy of the product. Product-related impurities include high molecular weight (HMW) and low molecular weight (LMW) impurities. HMW molecular species include, among other things, dimers (e.g., homodimers), oligomers, and higher-order aggregates. LMW molecular species have less positive charge than or similar surface charge to the product of interest and tend to elute together with or before the product of interest. LMW molecular species include, among other things, cleaved proteins, degraded proteins, and enzymatically cleaved proteins expressed during culture. These impurities may arise from structural heterogeneity that occurs during expression as a result of process conditions such as pH and shear. In some embodiments, bound impurities include microorganisms such as viruses, bacteria, and fungi. Such biocontaminations must be reduced or removed during downstream processing because their presence in the final drug substance or formulation renders the material unusable.

[0333] Frequent washing can control the accumulation of impurities and contaminants, which could be carried over from one cycle to the next and enter the product flow. Chromatographic media can be washed after every chromatographic batch in the manufacturing process. In some embodiments, at least one linear salt gradient non-denaturing washing step is performed after one or more cycles of the chromatographic column. Washing with at least a linear salt gradient as described herein after a cycle can reduce fouling, minimize degradation of the packed column, enable recycling of the chromatographic media, and / or extend the life of the chromatographic media. At least one linear salt gradient non-denaturing washing step can be combined with one or more denaturing washing steps and performed together or separately after one or more cycles, after one or more batches, and / or before storage. In some embodiments, a linear salt gradient washing step is performed after each cycle.

[0334] Linear salt concentration gradient buffer Some embodiments of this disclosure utilize a linear salt concentration gradient as a non-denaturing washing step. Specifically, the salt concentration gradient increases linearly from a low or no salt concentration to a higher salt concentration. In some embodiments, the linear salt concentration gradient is generated using at least two buffers (e.g., buffers 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the linear salt concentration gradient is generated using two buffers. In some embodiments, the linear salt concentration gradient is 0mM to 2M, or 0mM to 1.9M, or 0mM to 1.8M, or 0mM to 1.7M, or 0mM to 1.6M, or 0mM to 1.5M, or 0mM to 1.4M, or 0mM to 1.3M, or 0mM to 1.2M, or 0mM to 1.1M, or 0mM to 1M, or 0mM to 950mM, or 0mM to 900mM, or 0mM to 850mM. This includes increasing salt concentrations from M, or 0mM to 800mM, or 0mM to 750mM, or 0mM to 700mM, or 0mM to 650mM, or 0mM to 600mM, or 0mM to 550mM, or 0mM to 500mM, or 0mM to 450mM, or 0mM to 400mM, or 0mM to 350mM, or 0mM to 300mM, or 0mM to 250mM, or 0mM to 200mM. In some embodiments, any 0mM in the aforementioned range may be replaced with 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100mM.

[0335] In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM to 2 M. In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM to 1 M. In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM to 250 mM. In some embodiments, the linear salt concentration gradient includes an increase in salt concentration from 0 mM to 200 mM.

[0336] The linear salt concentration gradients described herein may use any salt suitable for desorbing impurities from the chromatographic medium. For example, in some embodiments, the linear salt concentration gradient is an NaCl gradient, a KCl gradient, a CaCl2 gradient, or a Na2SO4 gradient. In some embodiments, the linear salt concentration gradient is an NaCl gradient. In some embodiments, the linear salt concentration gradient is a KCl gradient. In some embodiments, the linear salt concentration gradient is a CaCl2 gradient. In some embodiments, the linear salt concentration gradient is a Na2SO4 gradient.

[0337] In some embodiments, the linear salt concentration gradient is an NaCl gradient, ranging from 0mM to 2M, or 0mM to 1.9M, or 0mM to 1.8M, or 0mM to 1.7M, or 0mM to 1.6M, or 0mM to 1.5M, or 0mM to 1.4M, or 0mM to 1.3M, or 0mM to 1.2M, or 0mM to 1.1M, or 0mM to 1M, or 0mM to 950mM, or 0mM to 900mM, or 0mM to 8 This includes increasing NaCl concentrations from 50 mM, or from 0 mM to 800 mM, or from 0 mM to 750 mM, or from 0 mM to 700 mM, or from 0 mM to 650 mM, or from 0 mM to 600 mM, or from 0 mM to 550 mM, or from 0 mM to 500 mM, or from 0 mM to 450 mM, or from 0 mM to 400 mM, or from 0 mM to 350 mM, or from 0 mM to 300 mM, or from 0 mM to 250 mM, or from 0 mM to 200 mM. In some embodiments, any 0 mM in the aforementioned ranges may be replaced with 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, the linear salt concentration gradient includes an increase in NaCl concentration from 0 mM to 2 M. In some embodiments, the linear salt concentration gradient includes an increase in NaCl concentration from 0 mM to 1 M. In some embodiments, the linear salt concentration gradient includes an increase in NaCl concentration from 0 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in NaCl concentration from 0 mM to 250 mM. In some embodiments, the linear salt concentration gradient includes an increase in NaCl concentration from 0 mM to 200 mM.

[0338] In some embodiments, the linear salt concentration gradient is a KCl gradient, ranging from 0mM to 2M, or 0mM to 1.9M, or 0mM to 1.8M, or 0mM to 1.7M, or 0mM to 1.6M, or 0mM to 1.5M, or 0mM to 1.4M, or 0mM to 1.3M, or 0mM to 1.2M, or 0mM to 1.1M, or 0mM to 1M, or 0mM to 950mM, or 0mM to 900mM, or 0mM to 8 This includes increases in KCl concentration from 50 mM, or from 0 mM to 800 mM, or from 0 mM to 750 mM, or from 0 mM to 700 mM, or from 0 mM to 650 mM, or from 0 mM to 600 mM, or from 0 mM to 550 mM, or from 0 mM to 500 mM, or from 0 mM to 450 mM, or from 0 mM to 400 mM, or from 0 mM to 350 mM, or from 0 mM to 300 mM, or from 0 mM to 250 mM, or from 0 mM to 200 mM. In some embodiments, any 0 mM in the aforementioned range may be replaced with 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, the linear salt concentration gradient includes an increase in KCl concentration from 0 mM to 2 M. In some embodiments, the linear salt concentration gradient includes an increase in KCl concentration from 0 mM to 1 M. In some embodiments, the linear salt concentration gradient includes an increase in KCl concentration from 0 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in KCl concentration from 0 mM to 250 mM. In some embodiments, the linear salt concentration gradient includes an increase in KCl concentration from 0 mM to 200 mM.

[0339] In some embodiments, the linear salt concentration gradient is a CaCl2 gradient, ranging from 0mM to 2M, or 0mM to 1.9M, or 0mM to 1.8M, or 0mM to 1.7M, or 0mM to 1.6M, or 0mM to 1.5M, or 0mM to 1.4M, or 0mM to 1.3M, or 0mM to 1.2M, or 0mM to 1.1M, or 0mM to 1M, or 0mM to 950mM, or 0mM to 900mM, or 0mM to 8 This includes increases in CaCl2 concentration from 50 mM, or from 0 mM to 800 mM, or from 0 mM to 750 mM, or from 0 mM to 700 mM, or from 0 mM to 650 mM, or from 0 mM to 600 mM, or from 0 mM to 550 mM, or from 0 mM to 500 mM, or from 0 mM to 450 mM, or from 0 mM to 400 mM, or from 0 mM to 350 mM, or from 0 mM to 300 mM, or from 0 mM to 250 mM, or from 0 mM to 200 mM. In some embodiments, any 0 mM in the aforementioned ranges may be replaced with 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, the linear salt concentration gradient includes an increase in CaCl2 concentration from 0 mM to 2 M. In some embodiments, the linear salt concentration gradient includes an increase in CaCl2 concentration from 0 mM to 1 M. In some embodiments, the linear salt concentration gradient includes an increase in CaCl2 concentration from 0 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in CaCl2 concentration from 0 mM to 250 mM. In some embodiments, the linear salt concentration gradient includes an increase in CaCl2 concentration from 0 mM to 200 mM.

[0340] In some embodiments, the linear salt concentration gradient is a Na2SO4 gradient, ranging from 0mM to 2M, or 0mM to 1.9M, or 0mM to 1.8M, or 0mM to 1.7M, or 0mM to 1.6M, or 0mM to 1.5M, or 0mM to 1.4M, or 0mM to 1.3M, or 0mM to 1.2M, or 0mM to 1.1M, or 0mM to 1M, or 0mM to 950mM, or 0mM to 900mM, or 0mM to 8 This includes increasing Na2SO4 concentrations from 50 mM, or from 0 mM to 800 mM, or from 0 mM to 750 mM, or from 0 mM to 700 mM, or from 0 mM to 650 mM, or from 0 mM to 600 mM, or from 0 mM to 550 mM, or from 0 mM to 500 mM, or from 0 mM to 450 mM, or from 0 mM to 400 mM, or from 0 mM to 350 mM, or from 0 mM to 300 mM, or from 0 mM to 250 mM, or from 0 mM to 200 mM. In some embodiments, any 0 mM in the aforementioned range may be replaced with 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, the linear salt concentration gradient includes an increase in Na2SO4 concentration from 0 mM to 2 M. In some embodiments, the linear salt concentration gradient includes an increase in Na2SO4 concentration from 0 mM to 1 M. In some embodiments, the linear salt concentration gradient includes an increase in Na2SO4 concentration from 0 mM to 500 mM. In some embodiments, the linear salt concentration gradient includes an increase in Na2SO4 concentration from 0 mM to 250 mM. In some embodiments, the linear salt concentration gradient includes an increase in Na2SO4 concentration from 0 mM to 200 mM.

[0341] Suitable buffers for generating a salt concentration gradient, depending on the pH of the chromatographic operation, include, but are not limited to, acetate buffer, phosphate buffer, sulfate buffer, carbonate buffer, piperazine buffer, imidazole buffer, Tris buffer, MES buffer, and any combination thereof. In some embodiments, at least one buffer used to generate the salt concentration gradient has substantially the same composition as the post-loading rinse solution.

[0342] In some embodiments, the buffer used to generate the salt concentration gradient contains an acetate salt. In some embodiments, the buffer contains at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, It contains at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, at least 500 mM, at least 550 mM, at least 600 mM, at least 650 mM, at least 700 mM, at least 750 mM, at least 800 mM, at least 850 mM, at least 900 mM, at least 950 mM, or at least 1 M of acetate salt.

[0343] In some embodiments, each buffer used to generate the salt gradient contains an acetate salt. In some embodiments, each buffer contains at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, at least 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, and at least It also contains 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, at least 350 mM, at least 400 mM, at least 450 mM, at least 500 mM, at least 550 mM, at least 600 mM, at least 650 mM, at least 700 mM, at least 750 mM, at least 800 mM, at least 850 mM, at least 900 mM, at least 950 mM, or at least 1 M of acetate.

[0344] In some embodiments, at least one buffer used to generate the salt gradient contains 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1 M acetate. In some embodiments, each buffer used to generate the salt gradient contains 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1 M acetate.

[0345] In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 500 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 100 mM. In some embodiments, at least one buffer used to generate a salt concentration gradient contains acetate at a concentration in the range of 50 mM to 100 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 50 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 55 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 60 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 65 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 70 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of at least 75 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of at least 80 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of at least 85 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of at least 90 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of at least 95 mM.

[0346] In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 100 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 50 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 51 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 52 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 53 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 54 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 55 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 56 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 57 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 58 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 59 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 60 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 65 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 70 mM. In some embodiments, at least one buffer used to generate a salt gradient contains acetate at a concentration of 75 mM.In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of 80 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of 85 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of 90 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of 95 mM. In some embodiments, at least one buffer used to generate the salt gradient contains acetate at a concentration of 100 mM.

[0347] In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 500 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 100 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration in the range of 50 mM to 100 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 50 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 55 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 60 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 65 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 70 mM. In some embodiments, each buffer used to generate a salt gradient independently contains acetate at a concentration of at least 75 mM. In some embodiments, each buffer used to generate the salt gradient independently contains acetate at a concentration of at least 80 mM. In some embodiments, each buffer used to generate the salt gradient independently contains acetate at a concentration of at least 85 mM. In some embodiments, each buffer used to generate the salt gradient independently contains acetate at a concentration of at least 90 mM. In some embodiments, each buffer used to generate the salt gradient independently contains acetate at a concentration of at least 95 mM.

[0348] In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 500 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 100 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration in the range of 50 mM to 100 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 50 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 55 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 60 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 65 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 70 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 75 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of at least 80 mM. In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of at least 85 mM. In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of at least 90 mM. In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of at least 95 mM.

[0349] In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 100 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at concentrations of 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 50 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 51 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 52 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 53 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 54 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 55 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 56 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 57 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 58 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 59 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 60 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 65 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 70 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 75 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 80 mM. In some embodiments, each buffer used to generate a salt gradient contains acetate at a concentration of 85 mM.In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of 90 mM. In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of 95 mM. In some embodiments, each buffer used to generate the salt gradient contains acetate at a concentration of 100 mM.

[0350] In some embodiments, each buffer used to generate the salt gradient contains an equal amount of acetate. In some embodiments, each buffer used to generate the salt gradient has the same composition except for the amount of salt in the buffer.

[0351] In some embodiments, linear salt concentration gradient washing is performed at a pH similar to or compatible with the chromatographic operation. In some embodiments, washing is performed at a pH of at least 3.6. In some embodiments, the pH is in the range of 3.6 to 6.0. In some embodiments, the pH is in the range of 4.0 to 5.6. In some embodiments, the pH is in the range of 4.5 to 5.6. In some embodiments, the pH is in the range of 5.0 to 5.6. In some embodiments, the pH is in the range of 5.5 to 5.6. In some embodiments, the pH is at least 4.0. In some embodiments, the pH is at least 4.1. In some embodiments, the pH is at least 4.2. In some embodiments, the pH is at least 4.3. In some embodiments, the pH is at least 4.4. In some embodiments, the pH is at least 4.5. In some embodiments, the pH is at least 4.6. In some embodiments, the pH is at least 4.7. In some embodiments, the pH is at least 4.8. In some embodiments, the pH is at least 4.9. In some embodiments, the pH is at least 5.0. In some embodiments, the pH is at least 5.1. In some embodiments, the pH is at least 5.2. In some embodiments, the pH is at least 5.3. In some embodiments, the pH is at least 5.4. In some embodiments, the pH is about 5.5. In some embodiments, the pH is about 5.6. In some embodiments, the pH is 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.

[0352] In some embodiments, each buffer used to generate the salt gradient has the same pH.

[0353] In some embodiments, linear salt concentration gradient washing is performed with a conductivity substantially similar to that of the post-loading rinse buffer.

[0354] In some embodiments, a linear salt concentration gradient is generated using two buffers: buffer A and buffer B. In some embodiments, a linear salt concentration gradient is generated using buffers A and B with a gradient from buffer A 100% (buffer B 0%) to buffer A 0% (buffer B 100%). In some embodiments, a linear salt concentration gradient is generated using buffers A and B with a gradient from buffer A 90% (buffer B 10%) to buffer A 10% (buffer B 90%). In some embodiments, a linear salt concentration gradient is generated using buffers A and B with a gradient from buffer A 80% (buffer B 20%) to buffer A 20% (buffer B 80%).

[0355] In some embodiments, buffer A contains 50 mM acetate and 0 mM sodium chloride, with a pH of 5.0 ± 0.1. In some embodiments, buffer B contains 50 mM acetate and 500 mM sodium chloride, with a pH of 5.0 ± 0.1. In some embodiments, buffer A contains 50 mM acetate and 0 mM sodium chloride at a pH of 5.0 ± 0.1, and buffer B contains 50 mM acetate and 500 mM sodium chloride at a pH of 5.0 ± 0.1.

[0356] In some embodiments, buffer A contains 50 mM acetate and 20 mM sodium chloride, with a pH of 5.0 ± 0.1. In some embodiments, buffer A contains 50 mM acetate and 20 mM sodium chloride at a pH of 5.0 ± 0.1, and buffer B contains 50 mM acetic acid and 500 mM sodium chloride at a pH of 5.0 ± 0.1.

[0357] Length and slope of a linear salt concentration gradient The slope of a salt concentration gradient is determined by dividing the change in salt concentration (M) by the length of the gradient (volume of media or column volume ("MV")): M / MV. When implementing a salt concentration gradient, there is a trade-off between the slope of the gradient and the amount of buffer consumed. A steep slope results in faster impurity removal and lower buffering requirements, but can lead to larger pressure spikes during washing. In contrast, a gentle slope results in slower impurity removal and higher buffering requirements, but reduces the risk of pressure spikes.

[0358] In some embodiments, the slope of the linear salt concentration gradient is 0.07 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.065 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.0625 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.06 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.0575 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.055 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.0525 M salt / MV buffer or less. In some embodiments, the slope of the linear salt concentration gradient is 0.05 M salt / MV buffer or less.

[0359] In some embodiments, the gradient length is at least 7.0 MV of buffer (i.e., at least 7.0 times the volume of the medium (e.g., column volume ("CV")) of buffer passed through the chromatographic medium during linear salt concentration gradient washing). In some embodiments, the gradient length is at least 7.1 MV. In some embodiments, the gradient length is at least 8.0 MV.

[0360] In some embodiments, the gradient length is in the range of 7.0 MV to 8.0 MV. In some embodiments, the gradient length is in the range of 7.1 MV to 8.0 MV.

[0361] In some embodiments, the gradient length is 7.0MV, 7.1MV, 7.2MV, 7.3MV, 7.4MV, 7.5MV, 7.6MV, 7.7MV, 7.8MV, 7.9MV, or 8.0MV. In some embodiments, the gradient length is 7.0MV. In some embodiments, the gradient length is 7.1MV. In some embodiments, the gradient length is 7.2MV. In some embodiments, the gradient length is 7.3MV. In some embodiments, the gradient length is 7.4MV. In some embodiments, the gradient length is 7.5MV. In some embodiments, the gradient length is 7.6MV. In some embodiments, the gradient length is 7.7MV. In some embodiments, the gradient length is 7.8MV. In some embodiments, the gradient length is 7.9MV. In some embodiments, the gradient length is 8.0MV.

[0362] In some embodiments, the gradient length is greater than 8.0 MV. In some embodiments, the gradient length is 9 MV. In some embodiments, the gradient length is 10.0 MV.

[0363] In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / MV buffer, and the length of the gradient is at least 7.1 MV (e.g., at least 8 MV). In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.065 M salt / MV buffer, and the length of the gradient is at least 7.1 MV.

[0364] In some embodiments, the slope of the linear salt concentration gradient is less than or equal to 0.065 M salt / MV buffer, and the length of the gradient is 8 MV.

[0365] Denaturation washing A denaturation washing step, including a denaturation washing step with a uniform concentration, can reduce fouling, minimize degradation of packed columns, enable the recycling of chromatographic media, and / or extend the life of chromatographic media. The chromatographic media washing methods provided herein may further include one or more denaturation washing steps (e.g., a denaturation washing step with a uniform concentration) that, in addition to removing impurities, also reduce or remove contaminants that contribute to the biocontamination load.

[0366] In some embodiments, one or more denaturation washing steps (e.g., denaturation washing at a uniform concentration) are performed after each cycle of the chromatographic medium. In some embodiments, one or more denaturation washing steps (e.g., denaturation washing at a uniform concentration) are performed after two or more cycles of the chromatographic medium. In some embodiments, one or more denaturation washing steps (e.g., denaturation washing at a uniform concentration) are performed following a batch of chromatography. In some embodiments, one or more denaturation washing steps (e.g., denaturation washing at a uniform concentration) are performed before storage. One or more non-denaturation washing steps with a linear salt concentration gradient and one or more denaturation washings (e.g., denaturation washing at a uniform concentration) may be performed together or separately after one or more cycles (i.e., after the same or different cycles), after one or more batches, and / or before storage. In some embodiments, at least one denaturation washing step (e.g., denaturation washing at a uniform concentration) is performed in combination with (e.g., following) a non-denaturation linear salt concentration gradient washing step. In some embodiments, two or more denaturation washing steps (e.g., denaturation washing at a uniform concentration) are performed following a non-denaturation linear salt concentration gradient washing step. In some embodiments, at least one denaturation washing step (e.g., denaturation washing at a uniform concentration) is performed after two or more non-denaturation linear salt concentration gradient washing steps. In some embodiments, at least one denaturation washing step (e.g., denaturation washing at a uniform concentration) is performed after one or more batches in combination with (e.g., following) a non-denaturation linear salt concentration gradient washing step. In some embodiments, at least one denaturation washing step (e.g., denaturation washing at a uniform concentration) is performed in combination with (e.g., following) a non-denaturation linear salt concentration gradient washing step before storage.

[0367] The denaturation washing step, for example, the denaturation washing step at a uniform concentration, is generally carried out under harsher conditions than the non-denaturation linear salt concentration gradient washing step described herein. Therefore, care must be taken when selecting operating conditions, as the denaturation washing step can cause degradation of the chromatography medium. In some embodiments, sodium hydroxide is used in the denaturation washing step. In some embodiments, sodium hydroxide is used in the denaturation solution at concentrations ranging from 0.5 M to 1.5 M. In some embodiments, 0.5 M sodium hydroxide is used in the denaturation solution. In some embodiments, 1 M sodium hydroxide is used in the denaturation solution. In some embodiments, 1.5 M sodium hydroxide is used in the denaturation solution.

[0368] In some embodiments, the contact time (i.e., the number of media volumes of the modified solution / flow rate) for the modified washing step (e.g., the uniform concentration modified washing step) is up to 8 hours. In some embodiments, the contact time is up to 7 hours, up to 6 hours, up to 5 hours, up to 4 hours, up to 3 hours, up to 2 hours, or up to 1 hour. In some embodiments, the contact time is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours.

[0369] In some embodiments, up to five times the volume of the medium is passed through the chromatographic medium in the denatured solution. In some embodiments, up to four times the volume of the medium is passed through the chromatographic medium in the denatured solution. In some embodiments, up to three times the volume of the medium is passed through the chromatographic medium in the denatured solution. In some embodiments, up to one time the volume of the medium is passed through the chromatographic medium in the denatured solution.

[0370] In some embodiments, one, two, three, four, or five times the volume of the medium is passed through the chromatographic medium in a denatured solution. In some embodiments, one volume of the medium is passed through the chromatographic medium in a denatured solution. In some embodiments, two volumes of the medium are passed through the chromatographic medium in a denatured solution. In some embodiments, three volumes of the medium are passed through the chromatographic medium in a denatured solution. In some embodiments, four volumes of the medium are passed through the chromatographic medium in a denatured solution. In some embodiments, five volumes of the medium are passed through the chromatographic medium in a denatured solution.

[0371] In some embodiments, the modified solution is used in a washing process with a uniform concentration.

[0372] Storage of chromatography media After a desired number of cycles or batches, the chromatography medium can be prepared for short-term or long-term storage. In some embodiments, the chromatography medium is prepared for storage after the completion of one or more batches. In some embodiments, the chromatography medium is prepared for storage after one or more cycles. In some embodiments, the chromatography medium is prepared for storage by subjecting it to one or more cycles using a protein-free loading solution.

[0373] In some embodiments, the chromatographic medium is stored in a storage solution (e.g., a storage solution containing sodium hydroxide). In some embodiments, the storage solution contains sodium hydroxide at a concentration in the range of 0.1 M to 0.2 M. In some embodiments, the storage solution contains sodium hydroxide at a concentration of 0.1 M. In some embodiments, the storage solution contains sodium hydroxide at a concentration of 0.125 M. In some embodiments, the storage solution contains sodium hydroxide at a concentration of 0.150 M. In some embodiments, the storage solution contains sodium hydroxide at a concentration of 0.175 M. In some embodiments, the storage solution contains sodium hydroxide at a concentration of 0.2 M.

[0374] In some embodiments, at least twice the volume of the medium is applied to the chromatographic medium in the storage solution. In some embodiments, at least three times the volume of the medium is applied to the chromatographic medium in the storage solution. In some embodiments, at least four times the volume of the medium is applied to the chromatographic medium in the storage solution. In some embodiments, up to five times the volume of the medium is applied to the chromatographic medium in the storage solution.

[0375] In some embodiments, one, two, three, four, or five times the volume of the medium is used to apply the preservation solution to the chromatographic medium. In some embodiments, one volume of the medium is used to apply the preservation solution to the chromatographic medium. In some embodiments, two volumes of the medium are used to apply the preservation solution to the chromatographic medium. In some embodiments, three volumes of the medium are used to apply the preservation solution to the chromatographic medium. In some embodiments, four volumes of the medium are used to apply the preservation solution to the chromatographic medium. In some embodiments, five volumes of the medium are used to apply the preservation solution to the chromatographic medium.

[0376] Quality of the product Product quality monitoring can be optionally performed in real time, near real time, and / or offline for each step of the biomanufacturing process. In some embodiments, product quality is assessed by the presence or absence of clipping, degradation, deamidation, and aggregation in the purified product. The term “clipping” refers to the partial cleavage of an expressed protein, usually by proteolysis. The term “degradation” generally means that a large substance, such as a peptide or protein, is broken down into two smaller substances, one of which may be significantly larger than the other. The term “deamidation” refers to any chemical reaction in which the amide functional group of the side chain of an amino acid, typically such as asparagine or glutamine, is removed or converted to another functional group. The term “aggregation” refers, for example, to aggregates of proteins as high molecular weight (HMW) molecular species. A non-limiting example of product quality is the absence or significant reduction of high molecular weight species. In particular, product quality in non-limiting examples is a high molecular weight content ranging from 10% to 50%.

[0377] The quality of the product can be monitored and measured using commercially available equipment and reagents, employing techniques known to those skilled in the art. Such methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA) and gel electrophoresis for detecting impurities such as eluted protein A and host cell proteins, as well as quantitative polymerase chain reaction (PCR) for nucleic acids, such as quantitative PCR (qPCR). Capillary electrophoresis, including size exclusion high-performance liquid chromatography (SE-HPLC) and reductive capillary electrophoresis (rCE-SDS), can be used to measure high molecular weight species (e.g., aggregates) and low molecular weight species of the product, including monomers, fragments, and unassembled components. Charged variants can be evaluated using techniques such as cation exchange high-performance liquid chromatography (CEX-HPLC).

[0378] Additional chromatography operations In some embodiments of this disclosure, one or more chromatographic operations may be performed upstream and / or downstream of the frontal chromatography operation described above. In some embodiments, one polishing chromatography operation is performed in a mode exhibiting a nonlinear relationship between step yield and loading density after at least one affinity chromatography operation. In some embodiments, one polishing chromatography operation is performed in frontal mode after at least one affinity chromatography operation. In some embodiments, one or more additional polishing chromatography unit operations may be performed before or after the frontal chromatography unit operation. One or more additional polishing chromatography unit operations may be performed in binding and elution mode, flow-through mode, weak partition chromatography mode, overload mode, and / or frontal mode. In some embodiments, one or more polishing chromatography operations performed in flow-through or weak partition chromatography mode follow the frontal chromatography operation. In some embodiments, the cation exchange chromatography unit operation is performed in a mode exhibiting a nonlinear relationship between step yield and loading density. In some embodiments, the cation exchange chromatography unit operation is performed in frontal mode.

[0379] Non-limiting examples of chromatographic media suitable for further polishing chromatographic operations include ion exchange chromatography (IEX) media, including cation exchange chromatography (CEX) and anion exchange chromatography (AEX) media, multimodal or mixed-mode chromatography (MMC) media, hydrophobic interaction chromatography (HIC) media, and hydroxyapatite (HA) media.

[0380] In addition to chromatographic operations, other unit operations may be included in the method herein, but are not limited to filtration operations such as deep filtration or viral filtration, and viral inactivation operations including low pH and surfactant viral inactivation.

[0381] host cell The cell lines used in this disclosure (also called “cells” or “host cells”) are genetically engineered to express proteins of commercial or scientific interest. The cells may be suitable for the adhesion, monolayer and / or suspension culture, transfection, and expression of recombinant proteins, such as antibodies. The cells may be used in conjunction with, for example, batch culture, fed-batch culture, and perfusion or continuous culture methods. Such cells are typically cell lines obtained from or derived from mammals and can grow and survive when placed in either monolayer or suspension culture in a medium containing appropriate nutrients and / or other factors, such as those described herein. Typically, host cells are selected that can express and secrete proteins, or that can be molecularly engineered to express and secrete large quantities of specific proteins, more specifically, glycoproteins of interest, into the culture medium. The selection of suitable host cells for expressing recombinant proteins will depend on a variety of factors, including the desired expression level, polypeptide modifications desirable or required for activity (such as glycosylation or phosphorylation), and the ease of folding into biologically active molecules. In some embodiments of the methods disclosed herein, the host cell is a mammalian host cell.

[0382] Cell lines generally originate from lines resulting from primary cultures that can be maintained in culture without time constraints. Cells may contain cells introduced via expression vectors (constructs), such as plasmids containing a coding sequence or part thereof that encodes a protein for expression and production in a culture process, for example, transformation, transfection, infection or injection. Such expression vectors contain elements necessary for the transcription and translation of the inserted coding sequence. Expression vectors containing sequences encoding desired proteins and polypeptides, as well as appropriate transcription and translation regulatory elements, can be constructed using methods well known to those skilled in the art and carried out by those skilled in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, 4. th This information is found in either the edition of Cold Spring Harbor Press, Plainview, NY, or an earlier edition; FMAusubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or an earlier edition; and Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990. All of these references are incorporated herein for all purposes.

[0383] Suitable host cells include, but are not limited to, those commercially available from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Braunschweig, Germany) or the American Type Culture Collection (ATCC).

[0384] Examples of host cells include, but are not limited to, prokaryotes, yeasts, or higher eukaryotic cells. Examples of prokaryotic host cells include bacteria, such as Gram-negative or Gram-positive microorganisms, such as Enterobacteriaceae, such as Escherichia, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, and Serratia, such as Serratia marcescens. Examples include marcescans, Shigella, and Bacillus, such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas, and Streptomyces. In some embodiments, eukaryotic microorganisms, such as filamentous fungi or yeasts, are suitable cloning or expression hosts for recombinant polypeptides. Saccharomyces cerevisiae, or common baker's yeast, are among the most commonly used lower eukaryotic host microorganisms.However, genera such as Pichia (e.g., P. pastoris), Schizosaccharomyces pombe, Kluyveromyces, Yarrowia, Candida, Trichoderma reesia, Neurospora crassa, and Schwanniomyces (e.g., Schwanniomyces occidentalis) are not included. Aspergillus (occidentalis), as well as filamentous fungi, such as the genera Neurospora, Penicillium, Tolypocladium, and Aspergillus, several other genera, species, and strains such as A. nidulans and A. niger are generally available and useful here.

[0385] Vertebrate host cells are also suitable hosts for recombinant protein expression. Suitable mammalian cell lines for recombinant protein expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but are not limited to, Chinese hamster ovary (CHO) cells, e.g., CHOK1 cells (ATCC CCL61), DXB-11, DG-44 and Chinese hamster ovary cell / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216, 1980); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, (Graham et al., J. Gen Virol. 36:59, 1977); baby hamster kidney cell line (BHK, ATCC CCL 10); Mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251, 1980); Monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); Human cervical cancer cells (HELA, ATCC CCL 2); Canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); Human lung cells (W138, ATCC CCL 75); Human hepatocellular carcinoma cells (Hep G2, HB 8065); Mouse mammary cancer cells (MMT 060562, ATCC CCL 51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68, 1982); MRC Examples include 5 cells or FS4 cells; mammalian myeloma cells and several other cell lines. In some embodiments, host cells are selected from CHO cells.

[0386] In some embodiments, the host cell is a eukaryotic cell, such as a mammalian cell. The mammalian cell may be, for example, a human, rodent, or bovine cell line or cell lineage. Examples of such cells, cell lines, or cell lineages include, but are not limited to, the mouse myeloma (NSO) cell line, the Chinese hamster ovary (CHO) cell line, FIT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BF1K (baby hamster kidney cell), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLa, EB1, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the mammalian cell is a CHO cell line. In some embodiments, the mammalian cell is a CHO cell. In some embodiments, mammalian cells are selected from CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS cells, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN cells, and CHO-derived cells. In some embodiments, CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. Furthermore, CHO FUT8 knockout cells are, for example, Potelligent® CHOK1 SV (Lonza, Inc.). In some embodiments, eukaryotic cells may also be avian cells, cell lines, or cell lines such as EBx® cells, EB14, EB24, EB26, EB66, or EBv13.

[0387] CHO cells, including CHOK1 cells (ATCC CCL61), are widely used to produce complex recombinant proteins. In some embodiments, dihydrofolate reductase (DHFR)-deficient mutant cell lines (Urlaub et al., 1980, Proc Natl Acad Sci USA 77:4216-4220), DXB11, and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplified gene expression systems allow for high levels of recombinant protein expression in these cell lines (Kaufman RJ, 1990, Meth Enzymol 185:537-566). Glutamine synthase (GS) knockout CHOK1SV cell lines utilizing methionine sulfoximine (MSX) selection based on glutamine synthase (GS) are also included. Other suitable CHO host cells for use in the bioreactor of the manufacturing process of the present disclosure include, but are not limited to, the following (ECACC acceptance numbers in parentheses): CHO (85050302), CHO (protein-free) (00102307), CHO-K1 (85051005), CHO-K1 / SF (93061607), CHO / dhFr- (94060607), CHO / dhFr-AC-free (05011002), and RR-CHOKI (92052129).

[0388] Large-scale production of commercially viable proteins can be carried out in suspension culture. Therefore, mammalian host cells used to produce the target protein can, but do not need to, be adapted for growth in suspension culture. Various host cells adapted for growth in suspension culture are known, including mouse myeloma NS0 cells and CLIO cells derived from CFIO-S, DG44, and DXB11 cell lines. Other suitable cell lines, but not limited to, mouse myeloma SP2 / 0 cells, baby hamster kidney BF1K-21 cells, human PER.C6® cells, human fetal kidney F1EK-293 cells, and cell lines derived from or manipulated from any of the cell lines disclosed herein.

[0389] In some embodiments, eukaryotic cells are, for example, yeast cells (e.g., Pichia species (e.g., methanol-assimilating yeast (Pichia pastoris), Pichia methanolica, Pichia kluyveri, and Pichia angusta)), Komagataella species (e.g., Komagataella pastoris, Komagataella pseudopastoris, or Komagataella phaffii)), Saccharomyces species (e.g., budding yeast (Saccharomyces cerevisae), Saccharomyces kluyveri, Saccharomyces ubarum) The cells are selected from lower eukaryotic cells such as *Cymbidium uvarum*, cells of the genus *Kluyveromyces* (e.g., *Kluyveromyces lactis*, *Kluyveromyces marxianus*), cells of the genus *Candida* (e.g., *Candida utilis*, *Candida cacaoi*, *Candida boidinii*), cells of the genus *Geotrichum* (e.g., *Geotrichum fermentans*), *Hansenula polymorpha*, *Yarrowia lipolytica*, or fission yeast (*Schizosaccharomyces pombe*). In some embodiments, eukaryotic cells are selected from methanol-assimilating yeast (Pichia pastoris) strains. Non-limiting examples of methanol-assimilating yeast (Pichia pastoris) strains include X33, GS115, KM71, KM71H, and CBS7435.

[0390] In some embodiments, eukaryotic cells are fungal cells (e.g., Aspergillus (e.g., A. niger, A. fumigatus, A. orzyae, A. nidula, etc.), Acremonium (e.g., A. thermophilum, etc.), Chaetomium (e.g., C. thermophilum, etc.), Chrysosporium (Chrysosporium) (e.g., C. thermophile), Cordyceps (e.g., C. militaris), Corynascus, Ctenomyces, Fusarium (e.g., F. oxysporum), Glomerella (e.g., G. graminicola), Hypocrea (Hyp ocrea (e.g., H. jecorina), Magnaporthe (e.g., M. orzyae), Myceliophthora (e.g., M. thermophile), Nectria (e.g., N. heamatococca), Neurospora (e.g., N. crassa), Penicillium Cells are selected from the genera (e.g., *T. llium*), *Sporotrichum* (e.g., *S. thermophile*), *Thielavia* (e.g., *T. terrestris*, *T. heterothallica*), *Trichoderma* (e.g., *T. reesei*), or *Verticillium* (e.g., *V. dahlia*).

[0391] In some embodiments, eukaryotic cells are insect cells (e.g., Sf9, Mimic® Sf9, Sf21, High Cells from Five (trademark) (BT1-TN-5B1-4), or BT1-Ea88 cells, etc., algal cells (e.g., Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, or Ochromonas, etc.), and plant cells (e.g., cells from monocots (e.g., maize, rice, wheat, or Setaria, etc.), or dicots (e.g., cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella, etc.) Cells are selected from (Patens) or (Arabidopsis, etc.).

[0392] To construct a host cell line (e.g., a mammalian cell line) engineered to express a target recombinant protein, one or more nucleic acids encoding the recombinant protein (or, in the case of a multichain protein, its components) are first inserted into one or more expression vectors. Useful nucleic acid regulatory sequences for expression vectors in mammalian cells include promoters, enhancers, and termination and polyadenylation signals. Optionally, secretion signal peptide sequences may also be encoded by the expression vector and operably linked to the target coding sequence, thereby causing the recombinant host cell to secrete the expressed protein, allowing for easier isolation of the recombinant protein from the cell if necessary. The vector may also include one or more selection marker genes to facilitate the selection of host cells into which the vector has been introduced. In some embodiments, vectors are used with protein fragment complementation assays using protein reporters such as dihydrofolate reductase (see, for example, U.S. Patent No. 6,270,964). Suitable mammalian expression vectors are known in the art and are commercially available.

[0393] Typically, a vector used in any host cell contains a sequence for maintaining the plasmid and a sequence for cloning and expressing the exogenous nucleotide sequence. Such a sequence typically includes the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional regulatory sequence and a translational regulatory sequence, a transcription termination sequence, a complete intron sequence containing a donor splice site and an acceptor splice site, a native or heterologous signal peptide sequence (leader sequence or signal peptide) for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a polynucleotide encoding the polypeptide to be expressed, and one or more selective marker elements. The vector may be constructed from a starting vector, such as a commercially available vector, and further elements may be obtained individually and ligated into the vector.

[0394] Culture method Batch culture, fed-batch culture, and perfusion culture are examples of various culture methods that can be used to produce the desired protein.

[0395] Batch culture is a discontinuous method in which cells are grown for a short period in a fixed volume of culture medium, and then the entire volume is harvested. Cultures grown using the batch method increase in cell density until they reach their maximum cell density, after which the viable cell density decreases as the culture medium components are consumed and metabolic by-products (such as lactic acid and ammonia) accumulate. Harvesting is typically performed when the maximum cell density (e.g., 5 × 10⁶ depending on the culture medium composition, cell line, etc.) is reached. 6 This process is performed when the cell density reaches a certain level (above or below cells / mL). While batch culture is the simplest culture method, the viable cell density is limited by nutrient availability, and the culture declines and production decreases once the cell density reaches its maximum. In batch culture, the production phase cannot be extended due to the accumulation of waste and the rapid decline of the culture due to nutrient depletion, and is usually about 3 to 7 days.

[0396] Fed-batch culture improves upon the batch process by providing bolus or continuous medium supply to replenish consumed media components. Because fed-batch culture receives additional nutrients throughout the operation, it results in higher cell densities (>10-30 × 10⁶ cells, depending on medium composition, cell line, etc.) compared to batch methods. 6Fed batch culture has the potential to achieve a higher bioproduct potency (cells / mL) and increased product potency. Unlike batch processes, by manipulating the feeding strategy and culture medium composition, a two-phase culture can be created and maintained, distinguishing between a cell growth phase (proliferation phase) to achieve the desired cell density and a period of cell growth cessation or slowing down (production phase). Therefore, fed batch culture has the potential to achieve a higher bioproduct potency compared to batch culture. Typically, the batch method is used during the growth phase and the fed batch method is used during the production phase, but a fed batch feeding strategy can be used throughout the entire process. However, unlike batch processes, the volume of the bioreactor becomes a limiting factor, and the amount of feed is limited. Similar to the batch method, the accumulation of metabolic byproducts can also lead to culture decline, which often limits the duration of the production phase to about 10-21 days. Fed batch culture is discontinuous, and harvesting is typically performed when metabolic byproduct levels or culture viability reach a predetermined level. Compared to batch culture without feeding, fed batch culture can produce a larger amount of recombinant protein. (For example, see U.S. Patent No. 5,672,502).

[0397] Perfusion culture offers potential improvements over batch and fed-batch methods by adding fresh medium during culture and simultaneously removing used medium. A typical perfusion culture begins with a batch culture startup lasting one or two days, followed by continuous, stepwise, and / or intermittent addition of fresh feeding medium to the culture, along with simultaneous removal of used medium, to retain cells and additional high molecular weight compounds (based on the filter's molecular weight cutoff value) such as proteins throughout the growth and production phases of the culture. Various methods, such as sedimentation, centrifugation, or filtration, can be used to remove used medium while maintaining cell density. Non-limiting examples of filtration methods include tangential flow filtration (TFF), e.g., recirculation flow filtration and alternating tangential flow (ATF) filtration. Alternating tangential flow is maintained by pressurizing the medium through a hollow fiber filter module. See, for example, U.S. Patent No. 6,544,424; Furey, 2002, Gen.Eng.News.22(7):62-63.

[0398] Perfusion can be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate can be less than or greater than the daily working volume. Cells are retained in the culture, and the used medium removed contains substantially no cells or significantly fewer cells compared to the culture. Recombinant proteins expressed by cell culture may also be retained in the culture.

[0399] Another type of perfusion culture, lean perfusion, operates at a high cell density during the growth phase and a low cell density during the production phase. As described in U.S. Provisional Patent Application No. 63 / 403,896, a low medium change rate is maintained throughout the culture to ensure the removal of waste by-products to avoid culture toxicity, while delivering sufficient nutrients via fresh medium to maintain essential cellular functions.

[0400] In a typical large-scale commercial cell culture strategy, biomass accounts for approximately one-third to more than one-half of the reactor volume, with a total volume of 40-90(+) × 10⁻¹⁰. 6 Cells / mL, e.g., approximately 40 × 10⁻⁶ 6 Cells / mL or approximately 50 × 10 6 The goal is to achieve a high cell density of cells / mL. In perfusion culture, >1 × 10 8 A very high cell density of cells / mL is achieved. A potential advantage of the perfusion process is that it allows for the maintenance of productive cultures for longer periods than batch or fed-batch cultures. However, maintaining long-term perfusion cultures requires increased preparation, use, storage, and disposal of the culture medium, especially in the case of high-cell-density cultures which also require more nutrients. Furthermore, high cell density can lead to problems in harvesting and downstream processes, such as issues associated with increased aeration, including more oxygen supply and more carbon dioxide removal, which may lead to problems during production, such as maintaining dissolved oxygen levels, and the need for more changes to foaming and defoaming strategies, as well as product loss due to the work required to remove excess cellular material, which may negate the benefit of increased titer due to increased cell volume.

[0401] Suitable culture conditions for mammalian cells, including temperature, dissolved oxygen content, and stirring speed, are known in the art and may vary depending on the stage or phase of cell culture. In some embodiments, the methods disclosed herein further include taking a sample during the cell culture process and evaluating the sample to quantitatively and / or qualitatively monitor the characteristics of recombinant proteins and / or the cell culture process. In some embodiments, the sample is monitored quantitatively and / or qualitatively using process analysis techniques. For example, dissolved oxygen levels may be monitored during the cell culture process using methods known in the art, such as basic chemical analysis (titration), electrochemical analysis (diaphragm electrode method), and photochemical analysis (fluorescence method).

[0402] It is desirable to have a controlled system that switches the physiological state of cells to a high-productivity state in which growth is restricted or stopped, using energy and substrates to allow cells to grow for a desired time or to a desired density while producing proteins, and then allowing the cells to use energy and substrates to produce recombinant proteins so that cell density can increase. For commercial-scale cell culture and the manufacture of biopharmaceuticals, the ability to restrict or stop cell growth and maintain cells in a growth-restricted or stopped state during the production stage is highly desirable. Such methods include, for example, temperature shifts, the use of chemical inducers of protein production, nutrient restriction or starvation, and cell cycle inhibitors, either alone or in combination. Exemplarily, a typical cell culture goes through a growth phase, which is a period of exponential growth in which cell density increases. During the growth phase, cells are cultured in a cell culture medium containing the necessary nutrients and additives under conditions (usually at a temperature of about 25-40°C in a humidified controlled atmosphere) in which optimal growth of a particular cell line is achieved. Cells are typically maintained for 1-8 days, e.g., 3-7 days, e.g., 7 days, during the growth phase. The length of the growth phase of a particular cell line can be determined by those skilled in the art, and is generally sufficient for the cells to proliferate to a viable cell density within the range of approximately 20% to 80% of the maximum viable cell density possible if the culture is maintained under growth conditions. Following the growth phase is a transitional phase in which exponential cell growth slows down and protein production begins to increase. This marks the start of the stationary phase, the production phase, in which cell density generally levels off and the titer of the product increases. During the production phase, the culture medium is usually replenished to support the continued production of recombinant protein.

[0403] In certain embodiments of the methods of this disclosure, culture conditions may be adjusted to accelerate the transition from the growth phase to the production phase of the cell culture. For example, the growth phase of the cell culture may occur at a higher temperature than the production phase of the cell culture. In some embodiments, the growth phase may occur at a first temperature of about 35°C to about 38°C, and the production phase may occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In some embodiments, a temperature shift from about 35°C to about 37°C to about 31°C to about 33°C may be used to accelerate the transition from the growth phase to the production phase of the culture. For example, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with the temperature shift, before and / or after the temperature shift, or in place of the temperature shift. If the inducers are added after the temperature shift, they may be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift.

[0404] Furthermore, any cell culture medium that can help promote the growth of suitable host cells in culture may be used. Typically, cell culture media contain buffers, salts, energy sources, amino acids, vitamins, and trace essential elements. Cell culture media are commercially available that may be further supplemented with other components to maximize cell proliferation, cell viability, and / or recombinant protein production in specific cultured host cells, including, among others, RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham F12 medium, Iskov Modified Dulbecco's medium, McCoy's 5A medium, Leibowitz L-15 medium, and serum-free media, such as the EX-CELL® 300 series, which can be obtained from American Type Culture Collection or SAFC Biosciences and other vendors. Cell culture media may be serum-free, protein-free, growth factor-free, and / or peptone-free. Cell culture media may also be eutrophicated by the addition of nutrients or other supplements, which may be used at concentrations higher than the usual recommended concentrations. In certain embodiments, the culture medium used in the methods of the present disclosure is a chemically defined medium, which refers to a cell culture medium in which all components have known chemical structures and concentrations. A chemically defined medium is typically serum-free and does not contain hydrolysates or animal-derived components.

[0405] Various culture media formulations can be used during the culture period to, for example, facilitate the transition from one stage (e.g., a growth stage or growth phase) to another stage (e.g., a production stage or production phase) and / or to optimize conditions during cell culture (e.g., a concentrated culture medium provided during perfusion culture). A growth culture medium formulation can be used to promote cell growth and minimize protein expression. A production culture medium formulation can be used to promote the production of the recombinant protein of interest and the maintenance of cells while minimizing the growth of new cells. A feed medium is typically a cell culture medium containing more concentrated components such as nutrients and amino acids that are consumed during the production stage of a cell culture. The feed medium can be used to supplement and maintain an active culture, particularly a culture operated in a fed-batch mode, a semi-perfusion mode, or a perfusion mode. Such a concentrated feed medium can contain most of the components of the cell culture medium, for example, about 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 12-fold, 14-fold, 16-fold, 20-fold, 30-fold, 50-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or even about 1000-fold their normal amounts.

[0406] In some embodiments of the methods of the present disclosure, mammalian cells are cultured for a predetermined period during which a recombinant protein is expressed and secreted by the mammalian cells. This period (i.e., the duration of the production stage of the cell culture) is at least 3 days, at least 7 days, at least 10 days, or at least 15 days. In certain embodiments, the duration of the production stage of the cell culture is about 7 days to 28 days, about 10 days to 30 days, about 7 days to 14 days, about 10 days to 18 days, about 3 days to 15 days, about 5 days to 8 days, about 12 days to 15 days, about 12 days to 18 days, or about 15 days to 21 days. In some embodiments, the duration of the production stage of the cell culture is 7 days, 8 days, 9 days, 12 days, 15 days, 18 days, or 21 days.

[0407] In some embodiments of the methods of the present disclosure, the viable cell density in the production stage is at least 100×10 5 cells / mL, for example, about 100×10 5 cells / mL to about 10×10 7 cells / mL, about 250×105 cells / mL ~ approx. 900×10 5 cells / mL, approximately 300×10 5 cells / mL~800×10 5 Cells / mL, or approximately 450 × 10⁻⁶ 5 cells / mL~650×10 5 Cell density is expressed as cells / mL. Cell density can be measured using a hemocytometer, Coulter counter, or automated cell analyzer (e.g., Cedex automated cell counter). Live cell density can be determined by staining the culture sample with trypan blue, which is taken up only by dead cells. The live cell density is then determined by counting the total number of cells, dividing the number of stained cells by the total number of cells, and taking the reciprocal.

[0408] bioreactor Cells can be cultured in suspension or in an adherent form bound to a solid culture medium. Cells can be established with or without microcarriers.

[0409] In some embodiments, cells are cultured in a bioreactor. The bioreactor may be a disposable container made of, for example, a plastic material, or a reusable container made of, for example, stainless steel. In some embodiments, the cell culture method of the present disclosure is carried out in a stainless steel bioreactor, for example, a built-in large stainless steel bioreactor.

[0410] In some embodiments, the volume of the bioreactor may range from 100 mL to 50,000 L. Unless otherwise specified, the bioreactor may be of any size as long as it is useful for cell culture. Typically, the bioreactor is sized appropriately for the volume of cell culture being cultured within it. In non-limiting embodiments, and unless otherwise indicated by the context, the bioreactor may be of at least 500 L, 1,000 L, 1,500 L, 2,000 L, 2,500 L, 5,000 L, 8,000 L, 10,000 L, 12,000 L, 18,000 L, 20,000 L or more, or any volume in between.

[0411] Internal conditions of the bioreactor, including but not limited to pH and temperature, can be controlled during the culture period. Those skilled in the art will be able to recognize and select a bioreactor suitable for use in the cell culture methods disclosed herein, based on the relevant considerations.

[0412] In some embodiments, the bioreactor may perform one or more (e.g., one, two, three, or all) of the following steps: supplying nutrients and / or carbon sources, injecting appropriate gases (e.g., oxygen), inflowing and outflowing cell culture media, separating gas and liquid phases, maintaining temperature, maintaining oxygen and CO2 levels, maintaining pH levels, stirring (e.g., agitation), and / or washing / sterilizing.

[0413] harvest The cell culture is harvested during and / or after the production stage. For example, the contents of a bioreactor may be partially harvested once or multiple times during the production stage. Alternatively, the contents of the bioreactor may be completely harvested at the end of the production stage. The harvesting operation completely or partially clarifies and / or purifies the target protein from at least one impurity found in the cell culture medium, such as residual cell culture medium, cells, cell debris, undesirable cell or medium components, and / or product-related and / or process-related impurities. Methods for harvesting recombinant proteins from suspended cell cultures are known in the art and include, but are not limited to, precipitation, e.g., acid precipitation; facilitated sedimentation, e.g., aggregation; separation by gravity; centrifugation; ultrasonic separation; filtration, including membrane filtration; ultrafiltration; microfiltration; tangential flow filtration; alternative tangential flow filtration; deep filtration; and synovial filtration (see, for example, U.S. Patent No. 9,371,554 and U.S. Patent No. 11,384,378).

[0414] The harvested cell culture medium (HCCF) can be stored in surge tanks, holding tanks, bags, or other containers suitable for supplying to downstream filtration or chromatography operations, and appropriate for infrastructure and / or process requirements. HCCF can also be supplied directly and continuously to downstream operations.

[0415] Affinity chromatography Affinity chromatography is generally used to initially separate a target protein from contaminants and impurities (such as product and / or process-related impurities) in a crude or clarified loading stream or pool. Affinity chromatography utilizes agents that bind and / or interact in some way with at least one desired protein, impurity, and / or contaminant.

[0416] In some embodiments, the harvested cell culture medium is subjected to affinity chromatography to isolate and concentrate the desired protein, for example, the desired protein having an Fc (fragment crystallization) component, from the harvested cell culture medium.

[0417] Non-limiting examples of affinity chromatography media include Staphylococcus aureus proteins, such as protein A, protein G, protein A / G, and protein L; substrate binding and capture mechanisms; antibody or antibody fragment binding and capture mechanisms; aptamer binding and capture mechanisms; and cofactor binding and capture mechanisms. Immobilized metal affinity chromatography (IMAC) can be used to capture proteins that have or have been engineered to have affinity for metal ions. Protein A affinity chromatography is typically used as the first-choice bulk purification of Fc (fragment, crystalline) region-containing proteins. Protein A ligands exhibit high selectivity for a wide range of proteins, providing high target protein yield and robust removal of process-related impurities. Suitable protein A methods and materials are widely known and available.

[0418] In some embodiments, the protein A chromatography medium is a high-volume chromatography medium such as MABSELECT® PRISMA. In some embodiments, the protein A chromatography medium is loaded to a loading density of 65 g / Lr.

[0419] An affinity chromatography operation may consist of a single independent skid operated one or more times to obtain a desired volume of product pool. An affinity chromatography operation may also include two or more independent skids operated simultaneously in parallel. Non-limiting examples of automated parallel chromatography systems are described in International Publication No. 2022 / 191971 and International Publication No. 2022 / 081939. In some embodiments, two or more affinity chromatography columns may be operated in series within a single skid. Various multi-column cycling strategies exist, such as closed-loop simulated mobile bed multi-column systems, sequential multi-column chromatography, and periodic countercurrent chromatography (PCC), an example thereof. In affinity chromatography operations using multiple skids, the same or similar chromatography media may be used.

[0420] Affinity chromatography, such as protein A affinity chromatography, is typically performed beforehand to clarify harvested cell culture media to a neutral pH. The affinity chromatography medium can be equilibrated with a suitable buffer before contact with the loading material containing the recombinant protein to be purified. The stream or pool containing the target product is loaded directly onto the affinity chromatography medium under conditions that promote protein binding.

[0421] Once the target product is loaded and bound, the chromatography medium is optionally rinsed with one or more rinse buffers before the elution step. The rinsing step can be performed to bind the target product that is on the column but not yet bound to the chromatography medium, to wash away loading material from the interstitial space, and to remove impurities bound to the chromatography medium and / or impurities present in the chromatography medium and impurities bound to the target product. Rinsing can also be used to prepare the column for elution. Multiple rinse buffers can be used depending on the purpose of rinsing and the number of steps. If multiple rinsing steps are used, the composition and / or concentration of the rinse buffer formulations may be the same or different as needed. Rinsing is performed at an appropriate pH, typically neutral pH, but may be performed at a higher or lower pH as needed or desired.

[0422] The bound target product can be eluted from the chromatographic medium by changing the buffer conditions. Various elution buffer formulations are known and used depending on the characteristics of the target product. Elution from affinity chromatography media is usually performed by a uniform concentration gradient under low pH conditions. In some embodiments, elution from affinity chromatography is performed by a linear pH gradient. In some embodiments, the affinity chromatography medium is a protein A affinity chromatography medium.

[0423] The cell culture harvest material may be loaded directly and continuously into one or more affinity chromatography skids via the outflow logistics from the harvesting operation, or it may be bulk loaded from a harvest hold tank or pool.

[0424] Virus inactivation Unit operations aimed at inactivating, reducing, and / or eliminating viral contaminants may include processes that mitigate viral risks through environmental manipulation and / or the use of filtration. Measures to reduce viruses are important for ensuring the safety of protein therapeutics and may be carried out one or more times throughout the downstream purification process. Viral contaminants can arise from a variety of sources, including the use of animal-derived reagents, exogenous viral contaminants in host cell lines, or system failures in GMP manufacturing sites. Viruses are classified into enveloped viruses and non-enveloped viruses. With enveloped viruses, the envelope allows the virus to identify, bind to, enter, and infect target host cells. Therefore, enveloped viruses are susceptible to inactivation methods. Various methods can be employed for virus inactivation, including thermal inactivation / pasteurization, UV and gamma irradiation, use of high-intensity broad-spectrum white light, addition of chemical inactivators and surfactants, and solvent / detergent treatment. Surfactants, such as those found in detergents, can solubilize membranes and therefore may be effective in specifically inactivating enveloped viruses; see, for example, International Publication No. 2020 / 190985.

[0425] Another method for achieving virus inactivation is incubation at a low pH (e.g., pH < 4). After low-pH virus inactivation, neutralization can be performed to readjust the pH of the acidic virus inactivation solution to a level suitable for subsequent downstream operations. Low-pH virus inactivation is typically performed after purification of harvested cell cultures by affinity chromatography, particularly protein A chromatography, which utilizes substrate-binding ligands derived from Staphylococcus aureus, because elution usually occurs at low pH. Bulk virus inactivation is performed in one or more holding tanks. A non-limiting process example of an automated two-tank low-pH inactivation and neutralization operation is described in International Publication No. 2022 / 099162. After low-pH virus inactivation, further filtration, such as deep filtration, may be performed for clarification and / or sterile filtration of the neutralized fluid.

[0426] Another method involves eluting the protein A affinity chromatography medium at a pH sufficient for viral inactivation. The acidified elution pool or flow acts as a loading feed for the polishing chromatography operation in frontal loading mode, and the loading time, rather than the flow between unit operations, is adjusted to meet the minimum residence time required for effective viral inactivation of the contents of the frontal chromatography elution product pool. This process eliminates the need for a holding tank and allows for a continuous, connected flow between chromatography unit operations. Furthermore, this process minimizes the amount of buffer and eliminates post-elution processing, resulting in a more rational, efficient, and robust process. These conditions are not only effective in inactivating the virus but can also increase product yield and reduce product-related impurities in the product pool from the frontal chromatography operation. The acidified elution pool or flow can be passed through one or more deep filters and / or sterile filters before frontal loading.

[0427] This method also has advantages over bulk pool or continuous inline virus inactivation strategies in that it supports a continuous process by maintaining a constant flow from one unit operation to another. A constant flow rate minimizes the time the product is exposed to tubes or other similar devices, reduces exposure to shear and other hydrodynamic forces, and allows for more accurate and flexible tracking of the time spent at low pH. In some embodiments, the residence time is at least 30 minutes. In some embodiments, the residence time is in the range of at least 60 minutes to at least 90 minutes. In some embodiments, the residence time is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 120 minutes, at least 180 minutes, at least 240 minutes, at least 300 minutes, or at least 360 minutes. In some embodiments, the residence time is at least 30 minutes. In some embodiments, the residence time is at least 45 minutes. In some embodiments, the residence time is at least 60 minutes. In some embodiments, the residence time is at least 75 minutes. In some embodiments, the residence time is at least 90 minutes. In some embodiments, the residence time is at least 120 minutes.

[0428] In some embodiments, the residence time from lowering the pH of the affinity chromatography eluate containing the target product to refurbishing the frontal loading product pool containing the target product is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, 42 hours, or 48 hours.

[0429] In other embodiments, the pH of the protein A chromatography effluent may also be adjusted inline between unit operations, rather than in a holding tank, to effectively inactivate the virus. This can be done using any of a variety of known methods, such as an inline mixer, a winding path, a coiled flow inverter, or other mechanisms for mixing and maintaining pH in a fluid flow.

[0430] The pH of the protein A eluate pool or effluent flow is adjusted with any suitable acid at a concentration suitable to meet the safety guidelines or regulations for biologics established by the relevant regulatory authority. In some embodiments, the pH of the protein A eluate pool or effluent flow is adjusted to a range of 3.0 to 4.0 before being used as a loading feed for frontal loading chromatography. In some embodiments, the pH is adjusted to a range of 3.3 to 4.0. In some embodiments, the pH is in the range of 3.3 to 4.0. In some embodiments, the pH is in the range of 3.4 to 4.0. In some embodiments, the pH is in the range of 3.5 to 4.0. In some embodiments, the pH is in the range of 3.6 to 4.0. In some embodiments, the pH is in the range of 3.7 to 4.0. In some embodiments, the pH is in the range of 3.8 to 4.0. In some embodiments, the pH is in the range of 3.9 to 4.0. In some embodiments, the pH is in the range of 3.3 to 3.6. In some embodiments, the pH is in the range of 3.4 to 3.6. In some embodiments, the pH is in the range of 3.5 to 3.6. In some embodiments, the pH is 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. In some embodiments, the pH is approximately 3.3. In some embodiments, the pH is approximately 3.6. In some embodiments, the pH is approximately 3.8. In some embodiments, the pH is approximately 4.0.

[0431] Virus filtration and UF / DF Non-enveloped viruses are more difficult to inactivate without risk against the manufactured protein and are removed by filtration. Viral filtration can be performed using PLAVONA® 20 and BioEx (Asahi Kasei, Chicago, IL), VIROSART® CPV, HC, HF and Media (Sartorius, Goettingen, Germany), VIRESOLVE® Pro and NFP (MilliporeSigma, Burlington, MA), Pegasus® Prime, VF DV20, DV 50 and SV4 (Pall Biotech, Port Washington, NY), and CUNO Zeta Plus VR (3M, St. Paul, Mn). Viral filtration is performed in one or more steps of the downstream process. Viral filtration may be performed prior to or following the ultrafiltration / diafiltration (UF / DF) operation.

[0432] The downstream process typically includes at least one ultrafiltration / diafiltration (UF / DF) operation for product concentration and buffer exchange. The UF / DF operation may be performed at one or more stages in the downstream process. Typically, the UF / DF operation is performed before bulk storage of the active pharmaceutical ingredient (API) to establish the API at the desired concentration and buffer formulation.

[0433] In some embodiments, the product pool obtained from the UF / DF operation is supplied directly to the filling / finishing operation. One or more stability-enhancing excipients may optionally be added directly to the UF / DF residue feed tank containing the formulated purified protein resulting in the formulated active pharmaceutical ingredient, or to the UF / DF eluate pool before filling / finishing. An example of a continuous operation from active pharmaceutical ingredient to formulation is provided in International Publication No. 2020 / 159838.

[0434] Filters for use in UF / DF operations are well-known and common in the art and are commercially available from many suppliers. A wide variety of UF / DF materials are available, including, but are not limited to, regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose SARTOCON® Slice, SARTOCON® ECO Hydrosart® (Sartorius, Goettingen, Germany), and polyethersulfone (PES) membrane Omega (Pall Corporation, Port Washington, NY).

[0435] protein The terms “polypeptide,” “protein,” and “product” are used interchangeably throughout this document and refer to molecules comprising two or more amino acid residues linked to one another by peptide bonds. Polypeptides, proteins, and products described herein include polymers having one or more deletions, insertions, and / or substitutions of amino acid residues in their native sequence, i.e., polypeptides, proteins, or products produced by naturally occurring non-recombinant cells or by genetically modified or recombinant cells, and include molecules having one or more deletions, insertions, and / or substitutions of amino acid residues in the amino acid sequence of a native protein. Polypeptides, proteins, or products provided herein also include amino acid polymers in which one or more amino acids are chemical analogs of corresponding naturally occurring amino acids and polymers. Polypeptides, proteins, or products disclosed herein may also include, but are not limited to, modifications such as glycosylation, lipid binding, sulfation, γ-carboxylation, hydroxylation, and ADP-ribosylation of glutamate residues.

[0436] Polypeptides, proteins, or products purified according to the chromatographic methods described herein may be of scientific and / or commercial interest, including protein-based therapeutic agents. Products of interest include, but are not limited to, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Polypeptides, proteins, and products of interest may be produced by cell lines (e.g., host cells) using the cell culture methods described herein and may be referred to as “recombinant proteins.” Expressed proteins may be produced intracellularly or secreted into a culture medium from which they can be recovered and / or collected. The terms “isolated protein” or “isolated recombinant protein” refer to a polypeptide or product of interest that has been purified by removing polypeptides, proteins, or other impurities that would interfere with therapeutic, diagnostic, preventive, research, and / or other uses. Products of interest include, but are not limited to, proteins that exert therapeutic effects by binding to targets, such as, for example, the targets listed herein, as well as derived targets, related targets, and their variants.

[0437] The products of interest may, but are not limited to, "antigen-binding proteins." An "antigen-binding protein" refers to a protein or polypeptide that contains an antigen-binding region or part that has affinity for another molecule (e.g., an antigen) to which it binds. Examples of antigen-binding proteins include, but are not limited to, antibodies, peptide bodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (e.g., single-chain variable fragments (scFv), double-chain (bivalent) scFv and IgGscFv (see, e.g., Orcutt et al., 2010, Protein Eng Des Sel 23:221-228)), heteroIgG (see, e.g., Liu et al., 2015, J Biol Chem 290:7535-7562), mutain, and XMAB® (Xencor, Inc., Monrovia, CA). This also includes bispecific T cell engager molecules (BITE® molecules), elongated bispecific T cell engagers, chimeric antigen receptors (CAR, CAR T), and T cell receptors (TCR).

[0438] In some embodiments, the desired product may contain a colony-stimulating factor such as granulocyte colony-stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, NEUPOGEN® (filgrastim) and NEULASTA® (pegfilgrastim).

[0439] Furthermore, erythropoiesis-stimulating agents (ESAs), such as EPOGEN® (epoetin alfa), ARANESP® (darbepoetin alfa), DYNEPO® (epoetin delta), MIRCERA® (methoxypolyethylene glycol-epoetin beta), HEMATIDE®, MRK-2578, INS-22, RETACRIT® (epoetin zeta), NEORECORMON® (epoetin beta), SILAPO® (epoetin zeta), BINOCRIT® (epoetin zeta) This also includes epoetin alfa, epoetin alfa HEXAL, ABSEAMED® (epoetin alfa), RATIOEPO® (epoetin theta), EPORATIO® (epoetin theta), BIOPOIN® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and their variants or analogues, as well as any of the aforementioned biosimilars.

[0440] In some embodiments, the product of interest is bound to one or more of the following, individually or in any combination: CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, CD174 CD proteins, HER receptor family proteins (e.g., HER2, HER3, HER4), and the EGF receptor EGFRvIII, cell adhesion molecules (e.g., LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, αv / β3 integrin), and growth factors (e.g., vascular endothelial growth factor "VEGF");VEGFR2), growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, growth hormone-releasing factor, parathyroid hormone, Müller inhibitor, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet-derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, epidermal growth factor (EGF), crypto, especially transforming growth factors including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5. TGF), insulin-like growth factor-I and-II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone induction factors, insulin and insulin-related proteins (including, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor-binding proteins), (coagulation and coagulation-related proteins, e.g., factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activator, e.g., urokinase and tissue protein) Rasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin and thrombopoietin receptors, colony-stimulating factor (CSF), other blood and serum proteins (including, but not limited to, M-CSF, GM-CSF, and G-CSF, albumin, IgE, and blood group antigens), receptors and receptor-related proteins (including, but not limited to, flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor), neurotrophic factors (including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophic factors). Trophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), relaxin A chain, relaxin B chain, and prorelaxin, interferons (e.g., including interferon-α, -β, and -γ), interleukins (IL), e.g., IL-1 to IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / IL-23, IL-2Ra, IL-1-R1, IL-6 receptor, IL-4 receptor and / or IL-13 receptor, IL-13RA2, or IL-17 receptor, IL-1RAP);This includes but is not limited to HIV enveloped virus antigen, lipoprotein, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor α and β, enkephalinase, viral antigens, BCMA, IgKappa, ROR-1, ERBB2, mesoserine, RANTES (an activation regulator normally expressed and secreted by T cells), mouse gonadotropin-related peptide, DNase, FR-α, inhibin, and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, and bone morphogenetic protein. (BMP), superoxide dismutase, surface membrane protein, disintegration factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP1-6, EPCAM, adresin, regulatory protein, immunoadhesin, antigen-binding protein, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit, B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, hepatitis C virus, mesoserine dsFv[PE38] conjugate, Legionella pneumophila (Ily), IFNγ, interferon-γ inducer protein 10 (IP10), IFNAR, T ALL-1, thymic interstitial lymphapoietin (TSLP), proprotein-converting enzyme subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet-specific (platelet glycoprotein IIb / IIIb (PAC-1), transforming growth factor β (TFGβ), zonaperusida sperm-binding protein 3 (ZP-3), TWEAK, platelet-derived growth factor receptor α (PDGFRα), sclerostin, and any of the aforementioned biologically active fragments or variants.

[0441] In some embodiments, the products of interest include absiximab, akapatamab, adalimumab, adekatumumab, aflibercept, alemtuzumab, arilocumab, anakinra, atacicept, basiliximab, belimumab, bemarituzumab, bevacizumab, biosozumab, blinatumomab, brentuximab vedotin, brodalumab, and cantuzumab meltan. Syn, canakinumab, cetuximab, certolizumab pegol, konatumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, efaveloukin alfa, epratuzumab, erenumab, etanercept, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab infliximab, ipilimumab, reldelimab B, Reticafusp, Lumiliximab, lxdkizumab, Mapatumumab, Motesanib diphosphate, Muromonab-CD3, Natalizumab, Nesilid, Nimotuzumab, Nivolumab, Ocrelizumab, Ofatumumab, Omalizumab, Oprelbequin, Ordecekimab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Pexelizumab, Ranibizumab, Rilo Examples include tumumab, rituximab, locatinlimab, romiplostim, romosozumab, rozibafusp alfa, sargamostim, tarlatamab, tezeperumab, tocilizumab, tositumomab, tiucetan, trastuzumab, ustekinumab, vedolizumab, bicilizumab, borosiximab, zanorimumab, and saltumumab, as well as any of the aforementioned biosimilars. [Examples]

[0442] The following embodiments are provided to illustrate various embodiments of the Disclosure and do not limit the Disclosure in any way. Those skilled in the art will readily understand that the Disclosure is well adapted to perform and achieve the purposes and benefits of the Disclosure, as well as the subject matter, purposes, and benefits inherent in this Specified Disclosure. Those skilled in the art will recognize the modifications and other uses in the embodiments that fall within the scope of the spirit of the Disclosure as defined by the claims.

[0443] Example 1. Non-denaturing washing method Non-limiting design requirements for effective column washing methods include robust impurity removal during washing and well-controlled column pressure. Impurity removal is crucial to prevent product carryover and resin fouling, extending resin life and achieving numerous reuse cycles. Well-controlled column pressure is critical for process safety and scalability. Column pressure control during washing after frontal chromatography is particularly important because this mode of operation results in a significant amount of bound product and / or bound impurities after product loading, leading to saturation or near-saturation of the column in typical product loading. Rapid desorption of bound components can result in highly concentrated boluses of product and / or impurities desorbing and moving through the column, causing spikes related to the overall pressure drop in the column (Δpressure = inlet pressure - outlet pressure).

[0444] The ability of a non-denaturing column washing method to effectively wash Frontal CEX chromatography columns while maintaining a stable column pressure was tested. CEX resin Eshmuno® CP-FT was used for all evaluations, and the following procedure was followed.

[0445] Loading preparation In each procedure, the loading material for frontal CEX chromatography consisted of a filtered neutralized virus inactivation pool (FVIP) containing monoclonal antibodies (mAb1, IgG2 antibodies). FVIP was prepared by purifying harvested cell culture medium with 50 mM sodium acetate, used as the protein A elution buffer, using protein A chromatography. Unless otherwise noted, the protein A pool underwent low-pH virus inactivation (VI) by titrating with 10% acetic acid to pH 3.6 ± 0.1 and neutralizing with 2 M Tris base to pH 5.0 ± 0.1. The acidified pool was held for a duration ranging from 60 to 90 minutes before neutralization, or neutralized immediately with minimal holding (i.e., mock VI). The VI procedure was performed at temperatures ranging from 15°C to 25°C. After VI, deep filtration was performed using a cellulose-based deep filter to produce the final FVIP, i.e., the loading for frontal loading CEX chromatography. The conductivity of the FVIP loading varied among these tests, as described below.

[0446] Column manipulation Figure 1 summarizes the basic operating sequence of Frontal CEX chromatography resin from equilibration to storage. The non-denaturing strip stage tested followed the loading and rinsing stage, and preceded regeneration washing with 1 M NaOH and column storage with 0.2 M NaOH. Table 1 provides further details on the tested loading and strip washing procedures. The initial evaluation criteria for the strip washing procedure were to control the pressure drop ΔP on the column during the strip stage and to achieve nearly complete washing over a predetermined time during the strip stage.

[0447] [Table 1]

[0448] Procedure 1 involved a strip washing step with a uniform concentration consisting of 10 mM acetate, 2 M sodium chloride, and pH 5.5. In this procedure, bench-scale screening was performed on a 17 cm bed height column, with short bursts of each phase including a 40 g / Lr column loading and a strip washing step of less than 1x the column volume. Due to the small loading volume in this screening run, a representative product pool was not recovered. However, excessive tailing of UV absorbance was observed during the strip washing step, indicating that desorption of bound products and impurities was slow even with small loading volumes, suggesting that the washing of the frontal chromatography CEX column in Procedure 1 may be inefficient.

[0449] Procedure 2 was used in a process robustness test that employed a strip washing step with a uniform concentration of 50 mM acetate, 1 M calcium chloride, and pH 5.0, testing various loading conditions over multiple cycles (Table 1). Procedure 2 had a moderate effect on washing the Frontal CEX resin, as observed from the UV absorbance peak reaching baseline during the strip washing step of each cycle. However, washing according to Procedure 2 resulted in an increase in column pressure during continuous column cycling.

[0450] Figure 2 shows the maximum ΔP observed during the strip washing stage in cycles 1, 2, 11, and 19 of the process robustness test. The cycles were performed with column loading of 750 g / Lr and material loading at pH 5.0 and conductivity of 5.3 mS / cm. While specific limitations on the operating pressure of any chromatography step vary based on process details such as column hardware, a robust washing procedure is expected to result in minimal pressure changes over consecutive cycles. As shown in Figure 2, the maximum ΔP increased by approximately twofold in cycle 19. The increase observed in a relatively small number of cycles at bench scale suggests that washing using procedure 2 may not enable reuse of chromatography resins at the number of cycles typically expected for reuse in large-scale production (e.g., 50 or more), increasing manufacturing costs and limiting its commercial-scale utility.

[0451] Step 3 consisted of a non-denaturing linear salt concentration gradient used to desorb and elute bound products and impurities at a well-controlled rate, minimizing pressure increases during the washing step. Furthermore, the high salt concentration achieved at the end of the gradient was designed to allow washing over multiple cycles. Sodium chloride was used to facilitate column regeneration with sodium hydroxide immediately after the strip washing step, a process sequence that could be risky if alternative salts such as calcium chloride were used due to potential precipitation in the hydroxide mixture.

[0452] Figure 3 shows the pressure trend of a typical chromatography, demonstrating that the strip washing pressure was well controlled. The linear salt concentration gradient in step 3 was also effective in controlling the column pressure over multiple cycles, as observed in a resin reuse study achieved over 100 cycles.

[0453] Figure 4 shows the maximum ΔP observed during the linear gradient strip washing phase in a representative control run performed with a 750 g / Lr loading and a linear velocity of 150 cm / hr. The relatively constant observed pressure indicates that the linear gradient washing in Step 3 is effective in minimizing fouling, which tends to increase in pressure over consecutive cycles. A further objective for column washing is to minimize potential product carryover between cycles. This was assessed by performing a simulated frontal CEX run over 100 cycles of Step 3 (with a "blank" loading consisting of equilibration buffer instead of the actual loading material), recovering the blank product pool, and examining trace amounts of protein via a fluorescence-based protein quantification assay (excitation / emission wavelengths: 280 / 335 nm). The measured protein carryover met the predetermined tolerance of ≤19.43 μg / mL, which corresponds to 1 / 1000 of the expected product concentration in a typical product pool, which is the standard tolerance for this test.

[0454] Example 2. Effect of virus inactivating acid titrator on HMW clearance on Frontal CEX column To investigate the effect of the acid titrator used for virus inactivation (VI) on high molecular weight (HMW) clearance across the entire column, a pilot-scale study was conducted using an ESHMUNO CP-FT® (EMD Millipore Corporation, Burlington, MA) column operated in frontal mode with high column loading. The acid titrator used for virus inactivation (VI) is often selected based on the matrix conditions of the preceding pool. For example, if the protein A chromatography elution buffer is sodium acetate, acetic acid is generally selected.

[0455] Table 2 summarizes the operational parameters used in the study. For each study condition, a filtration-neutralized virus inactivation pool (FVIP) containing mAb1 was prepared using one of two acid titrators and filtered using a cellulose-based depth filter. The FVIP was used as the loading feed for the CEX column. The criteria for starting and stopping the recovery of the product pool were determined by monitoring the UV absorbance at 280 nm.

[0456] Table 3 summarizes the HMW clearance observed for mAb1 pools when 1M formic acid or 10% acetic acid was used as the VI acid titrant. While the absolute HMW% level in the CEX pool did not vary significantly based on the choice of VI acid titrant, sample 2 (prepared with formic acid) was loaded with significantly higher CEX column loading compared to sample 1 (prepared with acetic acid). Therefore, the observation of comparable final HMW levels suggests that the use of formic acid allows for robust impurity clearance over a wider loading range, likely due to the lower FVIP conductivity achieved with formic acid compared to acetic acid. The relative increase in buffering capacity in the acetate-based product pool in VI is lower when formic acid is used, which in turn reduces the volume of base required to achieve the target pH during subsequent neutralization and the resulting FVIP conductivity. The reduced loading conductivity leads to stronger electrostatic interactions with the oppositely charged CEX resin, thereby potentially facilitating stronger binding to the resin and greater impurity removal, although the step yield is somewhat reduced due to greater product retention on the resin.

[0457] [Table 2]

[0458] [Table 3]

[0459] Example 3. Effect of virus inactivating acid titrator on viral clearance on a Frontal CEX column To ensure the safety of the product, the downstream purification process must demonstrate the ability to remove excess retroviral material across various steps for virus inactivation (e.g., by low pH) and removal (e.g., by chromatography or filtration). The viral clearance capability of ESHMUNO CP-FT® resin on a bench scale was characterized by loading the model virus, heterotropic mouse leukemia virus-associated virus (XMuLV), into FVIP and adding the added FVIP to CEX resin in high loading volumes. The FVIP loading material was obtained from a pilot-scale run using either 10% acetic acid or 1M formic acid as a virus inactivation titrator, followed by neutralization to pH 5 and filtration using a cellulose-based deep filter. Table 4 summarizes the details related to loading preparation and column operation.

[0460] The product pool fraction was recovered throughout the loading process, and viral titers were determined for the loading starting material, various fractions, and a pseudo-pool created to approximate the conditions of the product pool. XMuLV titers were measured by in-house qPCR assays. The viral log reduction (LRV) over unit operations was estimated based on loading and pseudo-pool titers as follows: LRV = log 10 (Total virus, Loading / Total virus, Pseudo-pool). Table 5 summarizes the results of these tests. In the case of mAb1, FVIP produced with 1M formic acid resulted in an additional XMuLV clearance exceeding 1.5LRV. The improvement in viral clearance capacity due to the formic acid production load may be due to relatively low conductivity, which promotes stronger binding of impurities such as foreign viruses.

[0461] [Table 4]

[0462] [Table 5]

[0463] Example 4. CEX chromatography yield and variable column volume Step yield is a critical performance metric for preparative chromatography, particularly for high-volume production materials requiring highly productive API processes. Chromatographic step yield can be significantly altered by column loading in operating modes where significant product binding to the column, such as binding and elution, and frontal chromatography are present. In frontal chromatography, since both monomers and impurities significantly bind to the chromatography resin, the yield is expected to increase monotonically with increasing loading. As the column is loaded beyond its saturation point and binding sites are occupied, the increasing fraction of loading material is recoverable because it flows through in the absence of free binding sites. This results in a general tendency for column loading to increase step yield, regardless of whether the process includes post-loading rinses to recover bound products. Figure 5 illustrates this tendency with pilot-scale and bench-scale process data for frontal CEX chromatography of mAb1 pools using Eshmuno® CP-FT resin. For all illustrated runs, loading was FVIP material (50 mM acetate buffer, pH 3.7 elution buffer) prepared using a Protein A pool as the starting material, followed by virus inactivation to pH 3.6 with 1 M formic acid, neutralization to pH 5.0 with 2 M Tris base, and deep filtration using a cellulose-based deep filter.

[0464] In an idealized large-scale manufacturing scenario, the loading mass to be purified across a column of a given size yields a near-optimal column load (or loading rate) with respect to step yield, using multiple column cycles to process the entire batch as needed. However, constraints on scaling the process to fit a given manufacturing site and / or variability in upstream yields often result in loading masses that do not allow for optimal column loading. This typically leads to either discarding excess product exceeding the maximum allowable loading (thus reducing yield) or processing the batch with additional cycles and using lower, suboptimal column loadings for each cycle (also reducing yield due to the aforementioned process tendencies). The risk of suboptimal yield due to upstream mass variability is higher when a given chromatography step requires several cycles to process a batch, as any additional cycles significantly reduce the loading required to process the same amount of material.

[0465] Column volume can be adjusted using column bed height to achieve well-controlled and near-optimal loading. Theoretically, column volume can also be adjusted via column diameter, but this strategy may not be practical in large-scale manufacturing facilities with limited hardware options. Furthermore, since column volume changes linearly with bed height and the square of the diameter, bed height can provide finer control over column volume.

[0466] To adjust column volume based on column bed height, the chromatography process step must be designed to achieve acceptable and consistent performance across a functional range of bed heights. Bench-scale tests were conducted to compare the performance of a Frontal CEX chromatography step using Eshmuno® CP-FT resin and mAb1 FVIP loading material at bed heights of approximately 10 cm and 15 cm. The mAb1 FVIP loading material, as with the FVIP loading described above, was generated from a pilot-scale run. Table 6 summarizes the relevant inputs and performance metrics (SE-HPLC HMW) used in this test. In addition to a test group operating 10 cm and 15 cm columns at standard flow rates for this step ("SFR", i.e., linear velocity during product loading and rinsing), a third group was included with a 10 cm bed height and a 33% reduced flow rate ("LFR" conditions, i.e., lower flow rate during product loading and rinsing) to match the residence time of the control group with a 15 cm bed height and a linear flow rate of 200 cm / hour. Typically, the resolution of chromatography improves as residence time and bed height increase. Therefore, conditions of 10 cm and SFR were expected to correspond to the worst-case scenario.

[0467] Removal of high molecular weight (HMW) molecular species is a key performance indicator of frontal CEX chromatography. HMW levels in SE-HPLC were tested not only at the loading volume (starting level) but also in a pseudo-pool (ending level) prepared by combining fractions collected during the loading process up to 1100 g / Lr. Under all three conditions, the HMW% level was less than 1.5%, representing a reduction of more than 50% compared to the starting level. These results suggest that the frontal CEX step for mAb1 can be effectively operated at various bed heights, including a 10 cm bed height, while maintaining standard process flow rates.

[0468] [Table 6]

[0469] Due to equipment size and pretreatment strategy constraints, given manufacturing facilities often present suboptimal choices regarding column diameter and the number of chromatography cycles required to process large volumes within a given time. Designing a more flexible process that allows column volume to be changed by bed height may limit the optimization of column loading that is inherent to the existing structure. Process modeling was also performed to predict the impact of varying column bed height on frontal CEX step yield. Figure 6 shows the results from this modeling, which predicts that, on average, the step yield can vary by up to approximately 7% based on a given set of equipment bed heights. As shown, the maximum predicted step yield is approximately 87% (achievable with maximum loading of the frontal CEX step), and this maximum can be achieved in different manufacturing scenarios by a wide range of target bed heights.

[0470] All documents or parts of documents cited herein, including but not limited to patents, patent applications, papers, books, and academic articles, are expressly incorporated herein by reference. The embodiments described herein may be combined with one or more other embodiments of the herein unless the context expressly indicates otherwise.

[0471] The subject matter disclosed is not intended to be limited in scope by any particular embodiment described herein, but rather is intended to serve as a non-limiting illustration of individual aspects of the disclosure. Functionally equivalent methods and components are within the scope of the disclosure. In fact, in addition to those shown and described herein, various modifications of the disclosed subject matter will be apparent to those skilled in the art from the above and the accompanying drawings. Such modifications are intended to be within the scope of the disclosed subject matter.

[0472] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for illustrative purposes only and are not intended to be exhaustive or restrictive in any way. Many variations and modifications will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terms used herein have been chosen to best describe the principles of the embodiments, their practical applications, or technical improvements to the art found in the market, and / or to enable those skilled in the art to understand the disclosed subject matter.

Claims

1. A method for washing chromatography media for reuse, A step of loading a composition containing a protein and at least one impurity into the chromatography medium, wherein the loading density exceeds the dynamic binding capacity of the chromatography medium to the protein. A step of recovering the fraction containing the aforementioned protein, A step of washing the chromatography medium using a linear salt concentration gradient. Methods that include...

2. The method according to claim 1, wherein the chromatography medium is a cation exchange (CEX) chromatography resin.

3. The method according to claim 1 or 2, wherein the chromatography medium is packed into a chromatography column.

4. The method according to any one of claims 1 to 3, wherein the loading density is at least 500 g / L-r.

5. The method according to any one of claims 1 to 4, wherein the loading density is in the range of 1000 g / L-r to 1500 g / L-r.

6. The method according to any one of claims 1 to 5, wherein the chromatography medium is loaded with at least one impurity in a saturated or near-saturated state.

7. The method according to any one of claims 1 to 6, wherein the at least one impurity comprises one or more high molecular weight species of the protein.

8. The method according to any one of claims 1 to 7, wherein the linear salt concentration gradient includes an increase in salt concentration from less than 50 mM to 500 mM.

9. The aforementioned linear salt concentration gradients are NaCl gradient, KCl gradient, CaCl ２ Gradient, or Na ２ SO ４ The method according to any one of claims 1 to 8, wherein the gradient is

10. The method according to any one of claims 1 to 9, wherein the linear salt concentration gradient is an NaCl gradient.

11. The method according to any one of claims 1 to 10, wherein the linear salt concentration gradient is generated using at least two buffers, and each of the at least two buffers used to generate the linear salt concentration gradient is independently selected from acetate buffer, phosphate buffer, Tris buffer, and 2-(N-morpholino)ethanesulfonic acid buffer.

12. The method according to claim 11, wherein each of the at least two buffer solutions used to generate the linear salt concentration gradient comprises an acetate.

13. The method according to claim 11 or 12, wherein the pH of each of the at least two buffers used to generate the linear salt concentration gradient is greater than 3.6 and less than 5.

6.

14. The method according to any one of claims 1 to 10, wherein the linear salt concentration gradient is generated by buffer A and buffer B, buffer A has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 0 mM or 20 mM sodium chloride, and buffer B has a pH of 5.0 ± 0.1 and contains 50 mM acetate and 500 mM sodium chloride.

15. The method according to claim 14, wherein the linear salt concentration gradient is generated using buffers A and B as a gradient from 100% buffer A (0% buffer B) to 0% buffer A (100% buffer B), or from 90% buffer A (10% buffer B) to 10% buffer A (90% buffer B), or from 80% buffer A (20% buffer B) to 20% buffer A (80% buffer B).

16. The method according to any one of claims 1 to 15, wherein the slope of the linear salt concentration gradient is less than or equal to 0.07 M salt / volume buffer solution.

17. The method according to any one of claims 1 to 16, wherein the length of the gradient is at least seven times the volume of the medium.

18. The method according to any one of claims 1 to 17, wherein the washing using the linear salt concentration gradient is non-modifying.

19. The method according to any one of claims 1 to 18, further comprising the step of washing the chromatography medium using a uniform concentration washing process using a denatured solution.

20. The method according to claim 19, wherein the modified solution contains sodium hydroxide.

21. The method according to any one of claims 1 to 20, wherein the method enables the reuse of the chromatography medium for multiple cycles.

22. The method according to any one of claims 1 to 20, wherein the method enables the reuse of the chromatography medium for at least 25 cycles.

23. A method for purifying a protein from a composition containing the protein and at least one impurity, A step of equilibrating the chromatography medium using an equilibration buffer, A step of loading the composition containing the protein and at least one impurity into the chromatography medium in frontal mode at a loading density of at least 500 g / L-r, A step of recovering the fraction containing the aforementioned protein, A step of passing a post-loading rinse solution, less than twice the volume of the medium, through the chromatography medium and adding the eluate to the fraction, A step of washing the chromatography medium using a linear salt concentration gradient, A step of washing the chromatography medium with a denatured solution and Methods that include...

24. The method according to claim 23, wherein fraction recovery is started at OD 0.5 in A280.

25. The method according to claim 23 or 24, wherein fraction recovery is stopped after passing the chromatographic medium through the post-loading rinse solution, which is one-fold or less of the volume of the medium.

26. The method according to any one of claims 23 to 25, wherein the equilibration buffer does not contain a salt.

27. The method according to any one of claims 23 to 26, wherein the modified solution is used in a washing process at a uniform concentration.

28. The method according to any one of claims 23 to 27, wherein the chromatography medium is a cation exchange (CEX) chromatography resin.

29. The method according to any one of claims 23 to 28, wherein the chromatography medium is packed into a chromatography column.

30. A method for improving the step yield of a frontal chromatography operation, A step to estimate the amount of protein in the loading solution, A step of determining the bed height of the frontal chromatography medium based on the target residence time, wherein the flow rate is a constant value in the range of 50 cm / hour to 250 cm / hour. Methods that include...

31. A method for improving the step yield of a frontal chromatography operation, A step to estimate the amount of protein in the loading solution, A step of determining the flow rate of the frontal chromatography operation based on the target residence time, A method comprising the following: the flow rate is in the range of 50 cm / hour to 250 cm / hour, and the bed height of the frontal chromatography medium is constant in the range of 5 cm to 30 cm.

32. The method according to claim 30 or 31, wherein the bed diameter of the frontal chromatography medium is kept constant.

33. The method according to any one of claims 1 to 32, wherein the protein is an antibody.

34. The method according to any one of claims 1 to 33, wherein the protein is an IgG2 antibody.

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