Protein purification method using chromatography

Abstract

To provide a combined chromatography process for the continuous purification of a protein of interest, in which anion exchange chromatography (AEX) and cation exchange chromatography (CEX) are operated in a flow-through mode without in-line adjustment of the chromatographic conditions, including pH and / or conductivity.SOLUTION: Provided is a method for purifying proteins using combined AEX-CEX chromatography in a flow-through mode under operating conditions without automation, engineering control and / or in-line adjustment between AEX and CEX. The AEX and CEX (or the CEX and AEX) can be directly connected.SELECTED DRAWING: None

Description

[Background technology]

[0001] Large-scale, efficient, and economical purification of proteins is one of the major hurdles to overcome in the current biotechnology and pharmaceutical industries. Generally, production and purification are first carried out by various “upstream” cell culture processes to produce the target protein. After production, “downstream” purification processes are used to separate and isolate the target protein from any undesirable contaminants that may also be present in the mixture produced in the upstream processes. Various upstream and downstream techniques can be found, for example, in Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Jagschies et al., 2017. Monoclonal antibodies (mAbs) and their derivative products (e.g., Fc fusion proteins, bispecific antibodies), which are specific subsets of proteins, play a crucial role in treating the most challenging human diseases due to the safety, efficacy, and high quality of these types of biologics. mAb production begins with protein expression in recombinant mammalian cultures. Subsequently, the clarified bulk is subjected to downstream purification (DSP). In downstream purification processes, protein A chromatography is typically used for capture, as described in Shukla et al., Downstream Processing Of Monoclonal Antibodies--Application Of Platform Approaches, J Chromatogr B Analyt Technol Biomed Life Sci, 848 (2007) 28-39, followed by one or two polishing steps. Cation exchange (CEX), anion exchange (AEX), hydrophobic interaction (HIC), and mixed chromatography are all used as polishing steps.

[0002] Current downstream purification is often carried out as a batch process with hold tanks between different unit operations (chromatography and filtration). Necessary buffer adjustments (e.g., pH or conductivity adjustments) are often required for each unit operation. Automation and in-line preparation of eluents have been reported to improve the purification process. Nevertheless, in-line preparation of buffer conditions is cumbersome and can increase operational costs, time, and skid footprint. [Overview of the project]

[0003] This disclosure relates to a combined chromatography process for the continuous purification of a target protein, operating AEX and CEX in flow-through mode without in-line adjustments to chromatographic conditions, including pH and / or conductivity. One aspect of this disclosure relates to the purification of a target protein using an AEX-CEX or CEX-AEX purification approach without in-line adjustments to either the pH or the conductivity of the purification conditions. Specifically, the approach uses tandem cation exchange chromatography (CEX) and anion exchange chromatography (CEX) in either the AEX-CEX or CEX-AEX sequence. In some embodiments, no adjustments are made to the pH or conductivity of the in-process pool between AEX and CEX or between CEX and AEX. Another feature of this disclosure is that the column conditions for AEX and CEX are processed together and operated as a single unit, which significantly reduces process cost, process operation time, and process skid footprint.

[0004] In some embodiments, the protein has an isoelectric point (pI) higher than approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In other embodiments, the protein has an isoelectric point (pI) lower than approximately 12.0, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, or 10.0.

[0005] In some embodiments, the protein has a pI of about 7.0 to about 11.0, about 7.0 to about 10.0, about 6.5 to about 10.5, or about 7.2 to about 9.6. In other embodiments, the protein has a pI of about 6.5, about 7.0, about 7.5, about 8.0, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, or about 11.0. In other embodiments, the protein has an isoelectric point (pI) lower than about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 5.9, about 5.8, or about 5.7.

[0006] In some embodiments, the protein has an isoelectric point (pI) higher than about 1.0, about 2.0, about 3.0, about 4.0, or about 5.0. In other embodiments, the protein has a pI of about 1.0 to about 6.5, about 2.0 to about 6.0, about 2.5 to about 6.5, or about 2.0 to about 5.8. In other embodiments, the protein has a pI of about 6.5, about 6.4, about 6.3, about 6.2, about 6.1, about 6.0, about 5.9, about 5.8, about 5.7, about 5.6, or about 5.5.

[0007] In some embodiments, combined chromatography includes adding a loading buffer and a chase buffer.

[0008] In some embodiments, the loading buffer has a pH higher than approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, approximately 9.0, approximately 9.5, or approximately 10.0. In other embodiments, the loading buffer has a pH of approximately 6.0 to approximately 11.0, approximately 6.5 to approximately 11.0, approximately 7.0 to approximately 10.5, approximately 7.0 to approximately 10.0, approximately 7.0 to approximately 9.5, approximately 7.0 to approximately 9.0, approximately 7.0 to approximately 8.5, approximately 7.0 to approximately 8.0, or approximately 7.0 to approximately 7.5. In other embodiments, the loading buffer has a pH of approximately 6.5, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0, 10.5, 11.0, or 11.5.

[0009] In some embodiments, the chase buffer has a pH higher than approximately 6.0, approximately 6.5, approximately 7.0, approximately 7.5, approximately 8.0, approximately 8.5, or approximately 9.0. In other embodiments, the chase buffer has a pH of approximately 6.0 to approximately 11.0, approximately 6.5 to approximately 10.0, approximately 7.0 to approximately 11.0, approximately 7.0 to approximately 10.0, approximately 7.0 to approximately 9.0, approximately 7.0 to approximately 8.0, approximately 7.0 to approximately 8.5, approximately 7.0 to approximately 9.5, approximately 6.5 to approximately 8.5, or approximately 6.5 to approximately 7.5. In other embodiments, the chase buffer has a pH of approximately 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9.0.

[0010] In some embodiments, the chase buffer has the same or a different pH as the loading buffer.

[0011] In some embodiments, the loading buffer has a conductivity of approximately 0.1 mS / cm to approximately 9.0 mS / cm, approximately 0.5 mS / cm to approximately 9.0 mS / cm, approximately 1.0 to approximately 9.0 mS / cm, approximately 2.0 to approximately 8.0 mS / cm, approximately 2.0 to approximately 7.0 mS / cm, approximately 2.0 to approximately 8.5 mS / cm, approximately 2.0 to approximately 7.5 mS / cm, or approximately 2.5 to approximately 7.0 mS / cm. In other embodiments, the loading buffer has a conductivity of approximately 0.1 mS / cm, approximately 0.5 mS / cm, approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, or approximately 9.0 mS / cm.

[0012] In some embodiments, the loading buffer has a conductivity of approximately 6.0 mS / cm to approximately 15.0 mS / cm, approximately 6.5 mS / cm to approximately 15 mS / cm, approximately 7.0 mS / cm to approximately 14.0 mS / cm, approximately 8.0 mS / cm to approximately 13.0 mS / cm, approximately 9.0 mS / cm to approximately 12.0 mS / cm, or approximately 9.0 mS / cm to approximately 9.5 mS / cm. In other embodiments, the loading buffer has a conductivity of approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10.0 mS / cm, approximately 10.5 mS / cm, approximately 11.0 mS / cm, approximately 11.5 mS / cm, approximately 12.0 mS / cm, approximately 12.5 mS / cm, approximately 13.0 mS / cm, approximately 13.5 mS / cm, approximately 14.0 mS / cm, approximately 14.5 mS / cm, or approximately 15.0 mS / cm.

[0013] In some embodiments, the chase buffer has a conductivity of approximately 0.1 mS / cm to approximately 9.0 mS / cm, approximately 0.5 mS / cm to approximately 9.0 mS / cm, approximately 1.0 to approximately 9.0 mS / cm, approximately 2.0 to approximately 8.0 mS / cm, approximately 2.0 to approximately 7.0 mS / cm, approximately 2.0 to approximately 8.5 mS / cm, approximately 2.0 to approximately 7.5 mS / cm, or approximately 2.5 to approximately 7.0 mS / cm. In other embodiments, the chase buffer has a conductivity of approximately 0.1 mS / cm, approximately 0.5 mS / cm, approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, or approximately 9.0 mS / cm. In other embodiments, the chase buffer has a conductivity of approximately 6.0 mS / cm to approximately 15.0 mS / cm, approximately 6.5 mS / cm to approximately 15.0 mS / cm, approximately 7.0 to approximately 15.0 mS / cm, approximately 8.0 to approximately 14.0 mS / cm, approximately 8.0 to approximately 13.0 mS / cm, approximately 8.0 to approximately 14.5 mS / cm, approximately 8.0 to approximately 13.5 mS / cm, or approximately 8.5 to approximately 13.0 mS / cm. In other embodiments, the chase buffer has a conductivity of approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, approximately 9.0 mS / cm, approximately 9.5 mS / cm, approximately 10.0 mS / cm, approximately 10.5 mS / cm, approximately 11.0 mS / cm, approximately 11.5 mS / cm, approximately 12.0 mS / cm, approximately 12.5 mS / cm, approximately 13.0 mS / cm, approximately 13.5 mS / cm, approximately 14.0 mS / cm, approximately 14.5 mS / cm, or approximately 15.0 mS / cm.

[0014] In some embodiments, the conductivity of the chase buffer is higher, lower, or the same as that of the loading buffer. In some embodiments, the conductivity of the chase buffer is at least 0.1 mS / cm, at least 0.2 mS / cm, at least 0.3 mS / cm, at least 0.4 mS / cm, at least 0.5 mS / cm, at least 1.0 mS / cm, at least 1.5 mS / cm, at least 2.0 mS / cm, at least 2.5 mS / cm, at least 3.0 mS / cm, at least 3.5 mS / cm, at least 4.0 mS / cm, at least 4.5 mS / cm, or at least 5.0 mS / cm higher than that of the loading buffer.

[0015] In some embodiments, the mixture contains one or more impurities. In some embodiments, the impurities include host cell proteins (HCPs), DNA, high molecular weight proteins (HMWs), low molecular weight proteins (LMWs), residual protein A (rPAs), or any combination thereof.

[0016] In some embodiments, chromatography reduces the level of impurities. In some embodiments, combined chromatography reduces the HCP level to about 100 ppm or less, about 90 ppm or less, about 80 ppm or less, about 70 ppm or less, about 60 ppm or less, about 50 ppm or less, about 40 ppm or less, about 30 ppm or less, about 20 ppm or less, or about 10 ppm or less. In some embodiments, combined chromatography reduces the HMW level to about 1.0% or less, about 0.9% or less, about 0.8% or less, about 0.7% or less, about 0.6% or less, about 0.5% or less, about 0.4% or less, about 0.3% or less, about 0.2% or less, or about 0.1% or less. In some embodiments, combined chromatography reduces LMW levels to approximately 1.0% or less, approximately 0.9% or less, approximately 0.8% or less, approximately 0.7% or less, approximately 0.6% or less, approximately 0.5% or less, approximately 0.4% or less, approximately 0.3% or less, approximately 0.2% or less, or approximately 0.1% or less. In some embodiments, combined chromatography reduces DNA levels to approximately 20 pg / mL or less, approximately 18 pg / mL or less, approximately 16 pg / mL or less, approximately 14 pg / mL or less, approximately 12 pg / mL or less, approximately 10 pg / mL or less, approximately 8 pg / mL or less, approximately 6 pg / mL or less, approximately 4 pg / mL or less, or approximately 2 pg / mL or less. In some embodiments, combination chromatography reduces the residual protein A (rPA) to approximately 6 ppm or less, approximately 5 ppm or less, approximately 4 ppm or less, approximately 3 ppm or less, approximately 2 ppm or less, approximately 1 ppm or less, approximately 0.8 ppm or less, approximately 0.6 ppm or less, approximately 0.5 ppm or less, approximately 0.4 ppm or less, approximately 0.3 ppm or less, approximately 0.2 ppm or less, or approximately 0.1 ppm or less.

[0017] A method for reducing or removing a buffer migration peak after a loading step in chromatography is also described, the method comprising adding a loading buffer and a chase buffer during chromatography for purifying a protein in a mixture, wherein the chase buffer has a higher conductivity than the loading buffer. In some embodiments, the chromatography is AEX chromatography, CEX chromatography, a combination of AEX and CEX chromatography, or a combination of CEX and AEX chromatography. In some embodiments, the chromatography is in flow-through mode. In some embodiments, the loading buffer has a conductivity of about 3.0 mS / cm to about 6.0 mS / cm, and the chase buffer has a conductivity of about 6.0 mS / cm to about 12.0 mS / cm. In some embodiments, the conductivity of the chase buffer is at least 0.5 mS / cm, at least 1.0 mS / cm, at least 1.5 mS / cm, at least 2.0 mS / cm, at least 2.5 mS / cm, at least 3.0 mS / cm, at least 3.5 mS / cm, at least 4.0 mS / cm, at least 4.5 mS / cm, or at least 5.0 mS / cm higher than the conductivity of the loading buffer. In certain embodiments, the loading buffer has a conductivity of 5.0 mS / cm, and the chase buffer has a conductivity of 6.0 mS / cm. In some embodiments, the loading buffer comprises sodium chloride, ammonium chloride, potassium chloride, sodium acetate, ammonium acetate, sodium sulfate, ammonium sulfate, ammonium thiocyanate, sodium citrate, sodium phosphate, and their potassium, magnesium, and calcium salts, or any combination thereof. In certain embodiments, the loading buffer comprises sodium acetate-Tris.In other embodiments, the chase buffer comprises sodium chloride, ammonium chloride, potassium chloride, sodium acetate, ammonium acetate, sodium sulfate, ammonium sulfate, ammonium thiocyanate, sodium citrate, sodium phosphate, and their potassium, magnesium, and calcium salts, or any combination thereof. In certain embodiments, the chase buffer comprises sodium acetate-Tris. In some embodiments, the chromatography comprises a column loaded with 50 g / L resin, 100 g / L resin, 150 g / L resin, 200 g / L resin, 250 g / L resin, 300 g / L resin, 350 g / L resin, 400 g / L resin, 450 g / L resin, or 500 g / L resin.

[0018] In some embodiments, chromatography is performed on Poros HS, Poros XS, carboxymethylcellulose, and Bakerbond ABX. TM Sulfopropyl and sulfonyl immobilized on agarose, MonoS, MiniS, Source 15S, 30S, SP SEPHAROSE TM CM SEPHAROSE TM, BAKERBOND Carboxy-Sulfon, WP CBX, WP Sulfonic, Hydrocell CM, Hydrocel SP, UNOsphere S, Macro-Prep High S, Macro-Prep CM, Ceramic HyperD S, Ceramic HyperD CM, Ceramic HyperD Z, Trisacryl M CM, Trisacryl LS CM, Trisacryl M SP, Trisacryl LS SP, Spherodex LS SP, DOWEX Fine Mesh Strong Acid Cation Resin, DOWEX MAC-3, Matrex Cellufine C500, Matrex Cellufine C200, Fractogel EMD SO3-, Fractogel EMD SE, Fractogel EMD COO-, Amberlite Weak and Strong Cation Exchangers, Diaion Weak and Strong Cation Exchangers, TSK Gel SP-5PW-HR, TSK Gel Includes CEX resins selected from SP-5PW, Toyopearl CM (650S, 650M, 650C), Toyopearl SP (650S, 650M, 650C), CM (23, 32, 52), SE (52, 53), P11, Express-Ion C and Express-Ion S, or any combination thereof.

[0019] In some embodiments, chromatography is performed using POROS HQ, POROS XQ, and Q SEPHAROSE TM Fast Flow, DEAE SEPHAROSE TM Fast Flow, SARTOBIND®Q, ANX SEPHAROSE TM 4 Fast Flow(high sub), Q SEPHAROSE TM XL, Q SEPHAROSE TMbig beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q SEPHAROSE TM high performance, Q SEPHAROSE TM XL, Sourse 15Q, Sourse 30Q, Resourse Q, Capto Q, Capto DEAE, Mono Q, Toyopearl Super Q, Toyopearl DEAE, Toyopearl QAE, Toyopearl Q, Toyopearl GigaCap Q, TS gel SuperQ, TS gel DEAE, Fractogel EMD TMAE, Fractogel EMD TMAE HiCap, Fractogel EMD DEAE, Fractogel EMD DMAE, Macroprep High Q, Macro-prep-DEAE, Unosphere Q, Nuvia Q, PORGS PI, DEAE Ceramic HyperD, Q Ceramic HyperD, or an AEX resin selected from any combination thereof.

[0020] In some embodiments, the protein is selected from an antibody or antibody fragment, a fusion protein, a native protein, a chimeric protein, or any combination thereof. In some embodiments, the antibody is an isotype selected from IgM, IgA, IgE, IgD, and IgG. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4.

[0021] In some embodiments, chromatography inactivates the virus. In some embodiments, the mixture is isolated by subjecting the starting mixture to affinity chromatography selected from Protein A affinity chromatography and Protein G affinity chromatography. In some embodiments, the starting mixture is selected from harvested cell culture fluid, cell culture supernatant, conditioned cell culture supernatant, cell lysate, and clarified bulk. In some embodiments, the starting material is derived from mammalian cell culture. In some embodiments, the starting mammalian cell culture is derived from Chinese hamster ovary (CHO) cells.

Brief Description of the Drawings

[0022] [Figure 1] Figure 1 shows a diagram of CEX-AEX or AEX-CEX combined polishing. The outlet of column 1 is directly connected to the outlet of column 2. There is no or no automation, engineering control, or regulation between these two columns.

[0023] [Figure 2] Figure 2 shows a representative chromatogram of CEX-AEX F / T combined polishing at a conductivity of 5 mS / cm and a pH of 7.2.

[0024] [Figure 3A] Figure 3A shows a chromatogram of CEX F / T loaded and traced at 5 mS / cm and pH 7.2. The loading buffer and the chase buffer have similar pH and conductivity using a sodium acetate-Tris buffer system. The post-loading peak induced by buffer transition was observed after the addition of the chase buffer, as measured by the absorbance at 280 nm.

[0025] [Figure 3B]Figure 3B shows the chromatogram of CEX F / T loaded at 5 mS / cm and pH 7.2, and tracked at 6 mS / cm and pH 7.2. Using a highly conductive buffer during tracking allows for efficient removal of peaks after loading. [Modes for carrying out the invention]

[0026] This disclosure provides a highly effective approach for removing contaminants during protein purification using affinity chromatography. Specifically, the approach uses tandem cation exchange chromatography (CEX) and anion exchange chromatography (AEX) in either AEX-CEX or CEX-AEX order. In some embodiments, the AEX and CEX are not modified in terms of pH or conductivity of the buffer between AEX and CEX or between CEX and AEX. Another feature of this disclosure is that the column conditions for AEX and CEX are processed together and done as a single unit, which significantly reduces process cost, process operation time, and process skid footprint.

[0027] As shown in the examples, this approach is effective in removing unwanted contaminants from a mixture of target proteins having a wide range of isoelectric points. By using such an AEX-CEX system, both the CEX and AEX processes can be improved simultaneously. In certain embodiments, the disclosure provides a method for purifying a target protein from a mixture containing the target protein and one or more contaminants (e.g., host cell proteins, DNA, residual protein A, etc.), wherein the CEX and AEX are operated in flow-through mode and the target protein does not strongly bind to either the CEX or the AEX. Using such a system to remove unwanted contaminants from a mixture using CEX and AEX enables extremely high throughput.

[0028] In certain embodiments, the Disclosure provides a method for purifying a target protein from a mixture containing the target protein and one or more contaminants. Possible contaminants include host cell proteins (HCPs), high molecular weight proteins (HMWs), low molecular weight proteins (LMWs), DNA, and / or residual protein A (rPA) from a previous protein A supplementation step. The Disclosure also provides a method for reducing a buffer migration post-loading peak, wherein the chromatographic process is interrupted when migrating from the loading step to a post-loading chase buffer for tracking the mixture from the column.

[0029] In certain embodiments, the disclosure provides a method for purifying antibodies. In certain embodiments, the mixture is derived from a harvested cell culture medium, cell culture supernatant, cell lysate, and clarified bulk.

[0030] I. Terminology As used herein, the term "and / or" should be interpreted as a specific disclosure of each of two specific features or components, with or without the other. Accordingly, as used herein, the term "and / or" in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (alone) and "B" (alone). Similarly, as used in phrases such as "A, B and / or C," the term "and / or" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] Whenever an aspect is described herein using the term "including," it is understood that other similar aspects are also provided, described using the terms "consisting of" and / or "essentially consisting of."

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide general dictionaries of many of the terms used herein.

[0033] Units, prefixes, and symbols are given in the form approved by the International System of Units (SI). Numerical ranges include the numerical values ​​that define the range. The headings provided herein may be obtained by referring to this specification as a whole, rather than being limitations on the various aspects of this disclosure. Thus, the terms defined immediately below are more fully defined by referring to this specification as a whole.

[0034] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives. The indefinite articles "a" or "an" used herein should be understood to refer to "one or more" of the listed or enumerated components.

[0035] The terms “approximately” or “essentially include” refer to a value or configuration that falls within the tolerance range of a particular value or configuration as determined by those skilled in the art, and this depends in part on how the value or configuration is measured or determined, i.e., the limits of the measurement system. For example, “approximately” or “essentially include” may mean within one or more standard deviations for each implementation in the art. Alternatively, “approximately” or “essentially include” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean a value up to one order of magnitude or up to five times. Where a particular value or configuration is provided in this application and claims, unless otherwise specified, the meaning of “approximately” or “essentially include” should be assumed to be within the tolerance range of that particular value or configuration.

[0036] Any concentration ranges, percentage ranges, ratio ranges, or integer ranges described herein should be understood to include, unless otherwise specified, any integer values ​​within the range described, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer).

[0037] As used herein, the term “target protein” is used in its broadest sense to include any protein (whether native or recombinant) present in the mixture for which purification is desired. Target proteins include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion proteins.

[0038] The term "clarification" refers to the process of removing particles. Clarification can reduce the burden on subsequent chromatography (e.g., AEX or CEX) during the purification process. In some cases, clarification is a method of removing colloids, lipids, DNA-RNA, residual cells, and other particles from a cell culture. Filtration may also be used, and may include depth filters. "Clarification bulk" refers to the mixture subjected to the clarification process.

[0039] The term "viral inactivation" refers to the process of removing infectious viral contaminants from a mixture. Currently, there are many different methods for inactivating infectious pathogenic viruses, such as thermal inactivation, organic solvent / surfactant (S / D) inactivation, pH inactivation, chemical inactivation, and / or ultraviolet irradiation inactivation.

[0040] The term "chromatography" refers to any technique for separating a target protein (e.g., an antibody) from other molecules (e.g., impurities) present in a mixture. Typically, the target protein is separated from other molecules (e.g., impurities) as a result of differences in the rates at which individual molecules of the mixture move through the stationary medium under the influence of the mobile phase, or as a result of binding and elution processes. The terms "matrix" or "chromatographic matrix" are used interchangeably herein and refer to any adsorbent, resin or solid phase that separates the target protein (e.g., a protein containing an Fc region, such as immunoglobulin) from other molecules present in the mixture during the separation process. Non-limiting examples include particulate, monolithic or fibrous resins, as well as membranes that can be placed in columns or cartridges. Examples of materials for forming the matrix include polysaccharides (e.g., agarose and cellulose); other mechanically stable matrices, such as silica (e.g., controlled-pore glass), poly(styrene-divinyl)benzene, polyacrylamide, ceramic particles, and any derivatives thereof. Typical matrix types suitable for the methods of this disclosure include cation exchange resins, affinity resins, anion exchange resins, or mixed resins. A "ligand" is a functional group that binds to a chromatographic matrix and determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interacting groups, hydrophilic interacting groups, thiophilic interacting groups, metallophilic groups, affinity groups, bioaffinic groups, and mixed groups (combinations thereof). Some preferred ligands that may be used herein include, but are not limited to, strong cation exchange groups, e.g., sulfopropyl, sulfonic acid; strong anion exchange groups, e.g., trimethylammonium chloride; weak cation exchange groups, e.g., carboxylic acids; weak anion exchange groups, e.g., N5N diethylamino or DEAE; hydrophobic interacting groups, e.g., phenyl, butyl, propyl, hexyl; and affinity groups, e.g., protein A, protein G, protein L. To facilitate understanding of this disclosure, certain terms are first defined.As used in this application, unless otherwise expressly provided herein, the following terms shall have the meanings set forth below. Further definitions are provided throughout this application.

[0041] The term "affinity chromatography" refers to a protein separation technique in which a target protein (e.g., a target protein containing an Fc region or an antibody) specifically binds to a ligand specific to that target protein. Such ligands are generally called biospecific ligands. In some embodiments, the biospecific ligand (e.g., protein A or a functional variant thereof) is covalently bound to the chromatography matrix material, and the target protein in the solution is accessible as the solution comes into contact with the chromatography matrix. The target protein generally retains its specific binding affinity to the biospecific ligand during the chromatography process, while other solutes and / or proteins in the mixture do not bind to the ligand to a degree that they can sense or specifically. Binding of the target protein to the immobilized ligand allows the target protein to remain specifically bound to the immobilized ligand on the solid phase material while interfering proteins or protein impurities pass through the chromatography matrix. The specifically bound target protein is then removed in its active form from the immobilized ligand under appropriate conditions (e.g., low pH, high pH, ​​high salt, competitive ligand, etc.) using an elution buffer, allowing it to pass through the chromatography column without the interfering proteins or protein impurities that were previously able to pass through the column. Any component can be used as a ligand for purifying its respective specific binding protein, such as an antibody. However, in various methods according to this disclosure, protein A is used as a ligand for a target protein containing an Fc region. The conditions for elution of the target protein (e.g., a protein containing an Fc region) from the biospecific ligand (e.g., protein A) can be readily determined by those skilled in the art. In some embodiments, protein G or protein L or their functional variants can be used as biospecific ligands. In some embodiments, the biospecific ligand, such as protein A, is used in a pH range of 5–9 for binding to the protein containing an Fc region, followed by washing or re-equilibrium of the biospecific ligand / target protein conjugate in a buffer with a pH of about 4 or less containing at least one salt, and subsequent elution.

[0042] The terms “purification,” “separation,” and “isolation,” as used interchangeably herein, refer to increasing the purity of a target protein from a composition or sample containing the target protein and one or more impurities. Typically, the degree of purity of the target protein is increased by removing (completely or partially) at least one impurity from the composition.

[0043] As used herein, the term "buffer" refers to a substance that, when present in a solution, increases the amount of acid or alkali that must be added to cause a unit change in pH. Buffers resist pH changes through the action of acid-base binding components. Buffers intended for use with biological reagents generally maintain a constant concentration of hydrogen ions so that the solution's pH remains within a physiological range. Conventional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases.

[0044] As used herein, the term "conductivity" refers to the ability of an aqueous solution to conduct electric current between two electrodes. In a solution, electric current flows by ion transport. Therefore, as the amount of ions present in the aqueous solution increases, the conductivity of the solution increases. The unit of measurement for conductivity is millisiemens per centimeter (mS / cm), and it can be measured using a conductivity meter.

[0045] As used herein, the terms “chromatography column” or “column” in relation to chromatography often refer to a container in the form of a cylinder or hollow pillar filled with a chromatography matrix or resin. The chromatography matrix or resin is a material that provides the physical and / or chemical properties used for purification.

[0046] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which an ionizable solute of interest (e.g., a target protein in a mixture) interacts with a solid-phase ion exchange material under appropriate pH and conductivity conditions via a countercharged ligand (e.g., covalently), thereby nonspecifically interacting with compounds that are more or less charged than solute impurities or contaminants in the mixture. Contaminating solutes in the mixture may be washed away from the ion exchange column or bound to or removed from the resin faster or slower than the solute of interest. Specifically, "ion exchange chromatography" includes cation exchange (CEX), anion exchange (AEX), and mixed-type chromatography.

[0047] A "cation exchange resin" or "cation exchange membrane" refers to a solid phase that is negatively charged and has free cations for exchange with cations in an aqueous solution that has passed over or through the solid phase. Any negatively charged ligand attached to a solid phase suitable for forming a cation exchange resin may be used, such as carboxylates, sulfonates, and others described below. Commercially available cation exchange resins include, for example, those with sulfonate-based groups (e.g., MonoS, MiniS, Source 15S and 30S, SP SEPHAROSE® Fast Flow, SP SEPHAROSE® High Performance, Capto S, Capto SP ImpRes (manufactured by GE Healthcare), TOYOPEARL® SP-650S and SP-650M (manufactured by Tosoh), MACRO-PREP® High S (manufactured by BioRad), Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS SP (manufactured by Pall Technologies)); those with sulfoethyl-based groups (e.g., FRACTOGEL® SE (manufactured by EMD), POROS® S-10 and S-20 (manufactured by Applied Biosystems)); and those with sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR (manufactured by Tosoh), POROS® HS-20, HS 50 and POROS® XS (manufactured by Life Technologies); those having a sulfoisobutyl-based group (e.g., FRACTOGEL® EMD SO3) -(EMD)); those with sulfoxyethyl-based groups (e.g., SE52, SE53 and Express-Ion S (Whatman)), those with carboxymethyl-based groups (e.g., CM SEPHAROSE® Fast Flow (GE Healthcare), Hydrocell CM (Biochrom Labs Inc.), MACRO-PREP® CM (BioRad), Ceramic HyperD CM, TRISACRYL® M CM, TRISACRYL® LS CM (Pall Technologies), Matrx CELLUFINE® C500 and C200 (Millipore), CM52, CM32, CM23 and Express-Ion C (manufactured by Whatman), TOYOPEARL® CM-650S, CM-650M and CM-650C (manufactured by Tosoh); those with sulfonic acid and carboxylic acid-based groups (e.g., BAKERBOND® carboxy-Sulfon (manufactured by JT Baker)); those with carboxylic acid-based groups (e.g., WP CBX (manufactured by JT Baker), DOWEX® MAC-3 (manufactured by Dow Liquid Separations), AMBERLITE® Weak Cation Exchangers, DOWEX® Weak Cation Exchanger, and DIAION® Weak Cation Exchangers (manufactured by Sigma-Aldrich) and FRACTOGEL® EMD COO-- (manufactured by EMD)); those with sulfonic acid-based groups (e.g., Hydrocell SP (manufactured by Biochrom Labs Inc.), DOWEX® Fine Mesh Strong Acid Cation Resin (manufactured by Dow Liquid Separations), UNOsphere S, WP Sulfonic (JTThis includes, but is not limited to, those having an orthophosphate-based group (e.g., P11 (Whatman)), such as Baker, SARTOBIND® S membrane (Sartorius), AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation, and DIAION® Strong Cation Exchanger (Sigma-Aldrich); or those having an orthophosphate-based group. Other cation exchange resins include carboxymethylcellulose, BAKERBOND ABX™, Ceramic HyperD Z, Matrex Cellufine C500, and Matrex Cellufine C200.

[0048] An "anion exchange resin" or "anion exchange membrane" refers to a solid phase that is positively charged and therefore has one or more positively charged ligands attached to it. Any positively charged ligand attached to a solid phase suitable for forming an anion exchange resin can be used, such as a quaternary amino group. Commercially available anion exchange resins include DEAE cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 (Applied Biosystems), SARTOBIND® Q (Sartorius), MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX, SEPHAROSE® Fast Flow, Q, SEPHAROSE® High Performance, QAE, SEPHADEX® and FAST Q, SEPHAROSE® (GE Healthcare), WP PEI, WP DEAM, WP QUAT (JT Baker), Hydrocell DEAE and Hydrocell QA (Biochrom Labs Inc.), UNOsphere Q, MACRO-PREP® DEAE and MACRO-PREP® High Q (Biorad), Ceramic HyperD Q, and ceramic HyperD DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q (manufactured by Pall Technologies), DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77, weak base anion (manufactured by Dow Liquid Separations), INTERCEPT® Q Membrane, Matrex CELLUFINE® A200, A500, Q500 and Q800 (manufactured by Millipore), FRACTOGEL® EMD TMAE, FRACTOGEL® EMDThis includes DEAE and FRACTOGEL® EMD DMAE (manufactured by EMD), AMBERLITE® weak strong anion exchangers type I and II, DOWEX® weak and strong anion exchangers type I and II, DIAION® weak and strong anion exchangers type I and II, DUOLITE® (manufactured by Sigma-Aldrich), TSK gel Q and DEAE 5PW and 5PW-HR, TOYOPEARL® SuperQ-650S, 650M and 650C, QAE-550C and 650S, DEAE-650M and 650C (manufactured by Tosoh), QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D or Express-Ion Q (manufactured by Whatman), and SARTOBIND® Q (manufactured by Sartorius Corporation, New York, USA). Other anion exchange resins include POROS XQ, SARTOBIND® Q, and Q SEPHAROSE TM XL, Q SEPHAROSE TM big beads, DEAE Sephadex A-25, DEAE Sephadex A-50, QAE Sephadex A-25, QAE Sephadex A-50, Q SEPHAROSE TM high performance, Q SEPHAROSE TM XL, Resource Q, Capto Q, Capto DEAE, Toyopearl GigaCap Q, Fractogel EMD TMAE HiCap, Nuvia Q or PORGS PI.

[0049] As used herein, the terms “flow-through” or “flow-through mode” refer to a common purification approach in which impurities are retained by the chromatographic process and typically bound to the resin in the column, thus being removed from the mixture during chromatography. The target protein is purified so that it does not bind to (or binds less strongly than) the chromatographic medium (typically the resin in the column) but instead flows through and is collected. After the elution of the target protein, any impurities bound to the column must be “striped” or removed so that the column can be regenerated for another chromatographic run. This approach differs from the “binding and elution” or “binding and elution mode” in which the target protein is retained on the column and impurities flow through the column. This process involves the specific elution of the target protein using various column conditions that prevent the target protein from binding to the chromatographic medium, typically the resin in the column.

[0050] As used herein, the term “impurities” is used in its broadest sense to cover undesirable components or compounds in a mixture. In cell cultures, cell lysates, or clarified bulk (e.g., clarified cell culture supernatant), impurities include, for example, host cell nucleic acids (e.g., DNA) and host cell proteins present in the cell culture medium. Host cell impurities include, but are not limited to, those produced naturally or recombinantly by the host cell, as well as proteins related to or derived from the target protein (e.g., proteolytic fragments) and other process-related impurities. In certain embodiments, impurity precipitates are separated from the cell culture by other means such as centrifugation, sterile filtration, depth filtration, and tangential flow filtration.

[0051] In some embodiments, the term “antibody” refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (hereinafter abbreviated as VH) and a heavy chain constant region (hereinafter abbreviated as CH). In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (hereinafter abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain (hereinafter abbreviated as CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The heavy chain may or may not have a C-terminal lysine. The term "antibody" may include bispecific or multispecific antibodies.

[0052] The “IgG antibodies” used herein, such as human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments, have the structure of naturally occurring IgG antibodies, i.e., the same number of heavy and light chains and disulfide bonds as naturally occurring IgG antibodies of the same subclass. For example, an IgG1, IgG2, IgG3, or IgG4 antibody may consist of two heavy chains (HC) and two light chains (LC), where the two HCs and LCs are linked in the same number and positions as the disulfide crosslinks that occur in naturally occurring IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (unless the antibody has mutated and the disulfide bonds have changed).

[0053] Immunoglobulins can originate from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are classified into subclasses in specific species; in humans, these are IgG1, IgG2, IgG3, and IgG4, while in mice, they are IgG1, IgG2a, IgG2b, and IgG3. Immunoglobulins, such as IgG1, exist in several allotypes, differing from each other by at most a few amino acids. "Antibodies" include, for example, both natural and non-natural antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies, and fully synthetic antibodies.

[0054] As used herein, the term “antigen-binding moiety” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. The antigen-binding function of an antibody has been shown to be performed by fragments of a full-length antibody. Examples of binding fragments included in the term “antigen-binding moiety” of an antibody include: (i) Fab fragments (fragments from papain cleavage) or similar monovalent fragments consisting of VL, VH, LC, and CH1 domains; (ii) F(ab')2 fragments (fragments from pepsin cleavage) or similar bivalent fragments containing two Fab fragments linked by disulfide bonds at a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single-strand antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) isolated complementarity-determining regions (CDRs); and (vii) combinations of two or more isolated CDRs that can be optionally bound by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker, which can be produced using recombinant DNA techniques to create a single protein chain in which the VL and VH regions pair up to form a monovalent molecule (single-stranded Fv, known as scFv; see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-stranded antibodies are also intended to be included in the term "antigen-binding moiety" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding moieties can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0055] As used herein, the term “recombinant human antibody” includes all human antibodies prepared, expressed, created or isolated by recombinant means, for example (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes of human immunoglobulin genes or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express antibodies, for example, transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, created or isolated by other means, including splicing human immunoglobulin gene sequences with other DNA sequences.

[0056] As used herein, "isotype" refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE antibodies).

[0057] Amino acids are represented herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides are represented by their commonly accepted one-letter codes.

[0058] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. As used herein, the term "protein" is intended to encompass a molecule composed of one or more polypeptides, which may, in some cases, be linked by bonds other than amide bonds. On the other hand, a protein can also be a single polypeptide chain. In this latter example, a single polypeptide chain may, in some cases, contain two or more polypeptide subunits that fuse together to form a protein. The terms "polypeptide" and "protein" also refer to products of post-expression modifications, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with amino acids that do not exist naturally. Polypeptides or proteins may originate from natural biological sources or be produced by recombinant techniques, but are not necessarily translated from a specified nucleic acid sequence. They may be produced by any means, including chemical synthesis.

[0059] As used herein, the terms “polynucleotide” or “nucleotide” are intended to encompass single nucleic acids and multiple nucleic acids and refer to isolated nucleic acid molecules or constructs, such as messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). In certain embodiments, polynucleotides include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (PNAs)).

[0060] The term “nucleic acid” refers to any one or more nucleic acid segments present in a polynucleotide, such as DNA, cDNA, or RNA fragments. Where applied to nucleic acids or polynucleotides, the term “isolated” refers to a nucleic acid molecule, DNA, or RNA taken from its natural environment; for example, a recombinant polynucleotide encoding an antigen-binding protein contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of this disclosure. Isolated polynucleotides or nucleic acids according to this disclosure further include such molecules produced synthetically. Polynucleotides or nucleic acids may also include regulatory elements, such as promoters, enhancers, ribosome-binding sites, or transcription termination signals.

[0061] The term "percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences across a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. Matching positions are any positions where identical nucleotides or amino acids are presented in both the target and reference sequences. Gaps presented in the target sequence are not counted because they are not nucleotides or amino acids. Similarly, gaps presented in the reference sequence are not counted because nucleotides or amino acids from the target sequence, not from the reference sequence, are counted. The percentage of sequence identity is calculated by determining the number of positions where identical amino acid residues or nucleic acid bases exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Sequence comparison and determination of percentage sequence identity between two sequences can be achieved using readily available software programs. Suitable software programs for both protein and nucleotide sequence alignment are available from various sources. One suitable program for determining percent sequence identity is bl2seq, which is part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs comparisons between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences.

[0062] The term "isoelectric point" or "pI" for a protein refers to the measured pH of a solution at which the protein has no net charge. When a protein is found at a pH equivalent to its pI, it has a net charge that is neutral overall. A protein with a pI lower than the pH of the solution has a net negative charge. Similarly, a protein with a pI higher than the pH of the solution has a net positive charge.

[0063] The term "loading buffer" refers to a buffer used to prepare a mixture or sample and load it into a chromatography unit.

[0064] The term "chase buffer" refers to a buffer used after the loading buffer to allow a mixture or sample to pass through the chromatography process.

[0065] The term "in-line adjustment" refers to any addition of a mixture or solution between AEX and CEX, or between CEX and AEX, to adjust the solution conditions or composition between chromatographic steps. In some embodiments, the AEX-CEX sequence is used, and no adjustment of pH and / or conductivity is made after the mixture leaves AEX and before it enters CEX. In other embodiments, the CEX-AEX sequence is used, and no adjustment of pH or conductivity is made after the mixture leaves CEX and before it enters AEX.

[0066] The term "HMW" refers to any one or more unwanted proteins present in a mixture. High molecular weight proteins can include dimers, trimers, tetramers, or other polymers. These species are often considered product-related impurities and can be either covalently or non-covalently bonded, and may consist of misfolded monomers, for example, hydrophobic amino acid residues exposed to polar solvents, which can cause aggregation.

[0067] The term "LMW" refers to any one or more unwanted proteins present in a mixture. Low molecular weight molecules are often considered product-related impurities and may include clipped species or half-molecules of compounds intended to be dimers (such as monoclonal antibodies).

[0068] The term "host cell protein" or HCP refers to undesirable proteins produced by host cells independently of the intended target protein. Undesirable host cell proteins can be secreted into the upstream cell culture supernatant. Undesirable host cell proteins can also be released during cell lysis. Cells used in upstream cell culture require proteins for growth, transcription, and protein synthesis, and these unrelated proteins are undesirable in the final pharmaceutical product.

[0069] The term “residual protein A” (rPA) refers to a protein originally found in the cell wall of the bacterium Staphylococcus aureus. Protein A is approximately 42 kDa and binds very strongly to the Fc portion of immunoglobulins; its use in antibody purification is well known in the art. Protein A has been widely used in the art for purification (Boyle et al., 1993; Hou et al. 1991). When applied to protein A, the terms “residual” or “rPA” refer to any remaining protein A present in a mixture because it is used in the further upstream purification of the target protein or antibody in the manufacturing process.

[0070] The term "buffer transition peak" refers to unwanted chromatographic elution caused by changes in conditions during chromatography. In some embodiments, the first buffer is a loading buffer used at the start of chromatography. In some embodiments, the second buffer is a chase buffer used to track the initially loaded mixture throughout the chromatography process. The buffer transition peak may appear during the transition from the use of the loading buffer to the use of the chase buffer. In other embodiments, the buffer transition peak may occur due to differences between the first buffer, e.g., the loading buffer, and the second buffer, e.g., the chase buffer.

[0071] Various aspects of this disclosure are described in further detail in the following subsections.

[0072] II. Purification method This disclosure relates to a method for purifying a target protein from a mixture, the method comprising performing a combination of cation exchange chromatography (CEX) and anion exchange chromatography (AEX) or a combination of AEX and CEX ("combined chromatography") in flow-through mode under operating conditions under which no changes to the operating conditions are made between AEX and CEX (or CEX and AEX). The absence of changes to the operating conditions between AEX and CEX (or CEX and AEX) includes, but is not limited to, automation, engineering control, and / or in-line adjustment. To avoid making changes between the AEX and CEX (or CEX and AEX) processes, in some embodiments, AEX and CEX (or CEX and AEX) may be in contact with each other, as shown in Figure 1. In other embodiments, AEX and CEX (or CEX and AEX) may not be directly connected to each other, but the operating conditions between AEX and CEX (or CEX and AEX) (e.g., pH and / or conductivity) remain unchanged, i.e., maintained in the same manner. The operating conditions may be changed by the use of buffers during chromatography. In some embodiments, the chromatography includes a loading buffer and / or a chase buffer.

[0073] In some embodiments, combined AEX and CEX (or CEX and AEX) chromatography columns are available in approximately 50 g / L resin, 60 g / L resin, 70 g / L resin, 80 g / L resin, 90 g / L resin, 100 g / L resin, 110 g / L resin, 120 g / L resin, 130 g / L resin, 140 g / L resin, and 150 g / L resin. fat, approx. 160g / L resin, approx. 170g / L resin, approx. 180g / L resin, approx. 190g / L resin, approx. 200g / L resin, approx. 210g / L resin, approx. 220g / L resin, approx. 230g / L resin, approx. 240g / L resin, approx. 250g / L resin, approx. 260g / L resin, approx. 270g / L resin, approx. 280g / L resin, approx. 290g / L resin, approx. 300g / L resin, approx. 3 10g / L resin, approx. 320g / L resin, approx. 330g / L resin, approx. 340g / L resin, approx. 350g / L resin, approx. 360g / L resin, approx. 370g / L resin, approx. 380g / L resin , approx. 390g / L resin, approx. 400g / L resin, approx. 410g / L resin, approx. 420g / L resin, approx. 430g / L resin, approx. 440g / L resin, approx. 450g / L resin, approx. 460g / L resin, approx. The columns are loaded with L-grade resin, approximately 470 g / L, 480 g / L, 490 g / L, 500 g / L, 510 g / L, 520 g / L, 530 g / L, 540 g / L, 550 g / L, 560 g / L, 570 g / L, 580 g / L, 590 g / L, or up to approximately 600 g / L. In some embodiments, combined AEX and CEX (or CEX and AEX) chromatography columns are loaded with up to approximately 300 g / L resin.

[0074] In some embodiments, if the pH of the buffer differs from the pI measurement of the protein, the target protein in this disclosure may be charged when placed in the buffer solution. When the target protein is found at a pH equivalent to its pI, it will have a net neutral charge overall. A target protein with a pI lower than the pH of the solution will have a net negative charge. Similarly, a target protein with a pI higher than the pH of the solution will have a net positive charge.

[0075] It is well known that the pH and ionic strength of the buffer are important for all forms of ion exchange chromatography, and therefore it is best to readjust the pH of the buffer after adjusting the salt concentration to ensure compatibility with the buffer's counterions. It is also known that the buffer's counterions should have the same charge as the resin. In particular, when two opposing chromatographic steps (e.g., AEX and CEX) are combined, it was expected that the pH and / or conductivity of AEX and CEX would be adjusted in between.

[0076] This method is based on the remarkable discovery that specific operating conditions for combined AEX and CEX (or CEX and AEX) chromatography can be maintained, for example, in flow-through mode, without adjustment between AEX and CEX (or vice versa).

[0077] In some embodiments, the operating conditions for a combination of AEX and CEX (or CEX and AEX) are set before the first chromatography and maintained throughout based on the pI of the target protein. In other embodiments, the operating conditions are set based on two different pIs, e.g., a medium to high pI and a low pI. In some embodiments, the operating conditions are set for a target protein having a medium to high pI, e.g., pI ≥ approximately 6.5, and for a target protein having a low pI, e.g., pI < 6.5.

[0078] In some embodiments, the target protein has a medium to high pI, which is higher than approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the target protein having a medium to high pI has pI ≥ 6.5 and pI ≤ approximately 12.0, approximately 11.9, approximately 11.8, approximately 11.7, approximately 11.6, approximately 11.5, approximately 11.4, approximately 11.3, approximately 11.2, approximately 11.1, approximately 11.0, approximately 10.9, approximately 10.8, approximately 10.8, approximately 10.7, approximately 10.6, approximately 10.5, approximately 10.4, approximately 10.3, approximately 10.2, approximately 10.1, or approximately 10.0. In some embodiments, the target protein having a medium to high pI has pI ≥ 6.5 and pI ≤ approximately 10.0, approximately 9.9, approximately 9.8, approximately 9.7, approximately 9.6, approximately 9.5, approximately 9.4, approximately 9.3, approximately 9.2, approximately 9.1, approximately 9.0, approximately 8.9, approximately 8.8, approximately 8.7, approximately 8.6, approximately 8.5, approximately 8.4, approximately 8.3, approximately 8.2, approximately 8.1, or approximately 8.0. In some embodiments, target proteins having a medium to high pI are approximately 7.0–11.0, 7.0–10.9, 7.1–10.9, 7.2–10.9, 7.2–10.8, 7.3–10.8, 7.3–10.7, 7.4–10.7, 7.4–10.6, 7.5–10.6, 7.5–10.5, 7.6–10.5, 7.6–10.4, and 7.7–10.4. The pI values ​​are approximately 7.7-10.3, 7.8-10.3, 7.8-10.2, 7.9-10.2, 7.9-10.1, 8.0-10.1, 8.0-10.0, 8.1-10.0, 8.1-9.9, 8.2-9.9, 8.2-9.8, 8.3-9.8, 8.3-9.7, 8.4-9.7, 8.4-9.6, 8.5-9.6, or 8.5-9.5. In other embodiments, the target protein has a pI of about 6.5, about 7.0, about 7.5, about 8.0, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, or about 11.0.

[0079] In some embodiments, the target protein having a moderate to high pI has a pI of about 7.0 to about 8.0, for example, about 7.6. In other embodiments, the target protein having a moderate to high pI has a pI of about 8.0 to about 9.0, for example, about 8.4. In some embodiments, the target protein having a moderate to high pI has a pI of 8.5 to 9.5, for example, about 8.9. In other embodiments, the target protein having a moderate to high pI has a pI of about 9.0 to about 10.0.

[0080] In some embodiments, the target protein has a low pI, which is lower than approximately 6.5, approximately 6.4, approximately 6.3, approximately 6.2, approximately 6.1, approximately 6.0, approximately 5.9, approximately 5.8, approximately 5.7, approximately 5.6, approximately 5.5, approximately 5.4, approximately 5.3, approximately 5.2, approximately 5.1, or approximately 5.0. In some embodiments, the target protein having a low pI is lower than 6.5 and higher than approximately 1.0, approximately 2.0, approximately 3.0, approximately 4.0, approximately 5.0, or approximately 5.5. In some minimal examples, the target protein with low pI has pI values ​​of approximately 1.0–6.5, 1.5–6.5, 2.0–6.5, 2.5–6.5, 2.5–6.0, 3.0–6.5, 3.0–6.0, 3.0–5.5, 3.5–5.5, or 3.5–5.0.

[0081] In some embodiments, the target protein has isoelectric points (pI) of approximately 4.8–6.5, 4.9–6.5, 4.9–6.4, 5.0–6.4, 5.0–6.3, 5.1–6.3, 5.1–6.2, 5.2–6.2, 5.2–6.4, or 5.3–6.4. In other embodiments, the target protein has a pI of approximately 5.0–6.0.

[0082] In certain embodiments, at least one of the operating conditions (e.g., pH) for target proteins having a medium to high pI and target proteins having a low pI may be the same. In other embodiments, the pH of the loading buffer and / or chase buffer may be the same for target proteins having a medium to high pI and target proteins having a low pI. In some embodiments, the pH of the loading buffer and / or chase buffer may differ for target proteins having a medium to high pI and target proteins having a low pI.

[0083] In some embodiments, the loading buffer for combination chromatography has a pH ≥ (greater than) of approximately 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. In some embodiments, the loading buffer has a pH of approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, It has a pH of approximately 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or higher than approximately 10.0. In some embodiments, the loading buffer is approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3 It has a pH of approximately 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0.

[0084] In some embodiments, the loading buffer for combination chromatography has pH values ​​of approximately 6.0 to 11.0, 6.5 to 11.0, 7.0 to 10.5, 7.0 to 10.0, 7.0 to 9.5, 7.0 to 9.0, 7.0 to 8.5, 7.0 to 8.0, 7.0 to 7.5, 7.0 to 7.4, 7.1 to 7.4, and 7.2 to 7.4.

[0085] In some embodiments, the loading buffer for combination chromatography has a pH of approximately 6.5, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0, 10.5, 11.0, or 11.5.

[0086] In some embodiments, the target protein has a high pI ranging from about 7.0 to about 8.0 (e.g., about 7.6), and the loading buffer for the protein has a pH ranging from about 7.0 to about 8.0, for example, about 7.4.

[0087] In another embodiment, the target protein has a high pI ranging from about 8.0 to about 9.0 (e.g., about 8.4), and the loading buffer for the protein has a pH ranging from about 7.0 to about 8.0, for example, about 7.2.

[0088] In some embodiments, the target protein has a high pI ranging from 8.5 to 9.5 (e.g., about 8.9), and the loading buffer for the protein has a pH ranging from about 7.5 to about 8.5, for example, about 8.0.

[0089] In another embodiment, the target protein has a high pI ranging from about 9.0 to about 10.0 (e.g., about 9.2), and the loading buffer for the protein has a pH ranging from about 7.0 to about 8.0, for example, about 7.2.

[0090] In another embodiment, the target protein has a low pI of about 5.0 to about 6.0, for example, about 5.8, and the loading buffer for the protein has a pH of about 6.5 to about 7.5, for example, about 7.0.

[0091] In other embodiments, the loading buffer and chase buffer for combined chromatography have the same operating conditions. In certain embodiments, buffer conditions, such as pH, are kept the same between the loading buffer and the chase buffer. In other embodiments, the buffer conditions between the loading buffer and the chase buffer are different.

[0092] In some embodiments, the chase buffer has a pH ≥ (greater than) of approximately 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. In other embodiments, the chase buffer has a pH ≥ (greater than) of approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, It has approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, approximately 9.4, approximately 9.5, approximately 9.6, approximately 9.7, approximately 9.8, approximately 9.9, approximately 10.0, approximately 10.1, approximately 10.2, approximately 10.3, approximately 10.4, approximately 10.5, approximately 10.6, approximately 10.7, approximately 10.8, approximately 10.9, or approximately 11.0.

[0093] In some embodiments, the chase buffer has a pH ≥ approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, and 7.8. It has approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, approximately 8.5, approximately 8.6, approximately 8.7, approximately 8.8, approximately 8.9, approximately 9.0, approximately 9.1, approximately 9.2, approximately 9.3, approximately 9.4, approximately 9.5, approximately 9.6, approximately 9.7, approximately 9.8, approximately 9.9, approximately 10.0, approximately 10.1, approximately 10.2, approximately 10.3, approximately 10.4, approximately 10.5, approximately 10.6, approximately 10.7, approximately 10.8, approximately 10.9 or approximately 11.0.

[0094] In some embodiments, the chase buffer has a pH of approximately 6.0 to 11.0, 6.5 to 11.0, 7.0 to 10.5, 7.0 to 10.0, 7.0 to 9.5, 7.0 to 9.0, 7.0 to 8.5, 7.0 to 8.0, 7.0 to 7.5, 7.0 to 7.4, 7.1 to 7.4, or 7.2 to 7.4. In some embodiments, the chase buffer has a pH of about 6.5, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.2, about 8.4, about 8.6, about 8.8, about 9.0, about 9.2, about 9.4, about 9.6, about 9.8, about 10.0, about 10.5, about 11.0, or about 11.5. In some embodiments, the chase buffer has a pH of about 6.0 to about 11.0, about 6.5 to about 10.0, about 7.0 to about 11.0, about 7.0 to about 10.0, about 7.0 to about 9.0, about 7.0 to about 8.0, about 7.0 to about 8.5, about 7.0 to about 9.5, about 6.5 to about 8.5, or about 6.5 to about 7.5.

[0095] In some embodiments, the target protein has a pI of about 7.0 to about 8.0, for example, about 7.6, and the loading buffer and chase buffer for the protein have the same pH of about 7.0 to about 8.0, for example, about 7.6.

[0096] In other embodiments, the target protein has a pI of about 8.0 to about 9.0, for example, about 8.4, and the loading buffer and chase buffer for the protein have the same pH of about 8.0 to about 9.0, for example, about 8.4.

[0097] In some embodiments, the target protein has a pI of 8.5 to about 9.5, for example, about 8.9, and the loading buffer and chase buffer for the protein have the same pH of 8.5 to about 9.5, for example, about 8.9.

[0098] In another embodiment, the target protein has a pI of about 9.0 to about 10.0, for example, about 9.2, and the loading buffer and chase buffer for the protein have the same pH of about 9.0 to about 10.0, for example, about 9.2.

[0099] In another embodiment, the target protein has a pI of about 5.0 to about 6.0, for example, about 5.8, and the loading buffer and chase buffer for the protein have the same pH of about 5.0 to about 6.0, for example, about 5.8.

[0100] In other embodiments, the operating conditions for target proteins having medium and high pI and target proteins having low pI may differ. In some embodiments, the conductivity for target proteins having medium and high pI and target proteins having low pI may differ. In some embodiments, the conductivity for proteins with low pI is higher than that for proteins with medium to high pI.

[0101] In some embodiments, the conductivity for loading buffers and / or chase buffers of a target protein having a medium to high pI is about 0.1 mS / cm to about 9.0 mS / cm, about 0.5 mS / cm to about 9.0 mS / cm, about 1.0 mS / cm to about 9.0 mS / cm, about 2.0 mS / cm to about 8.0 mS / cm, about 2.0 mS / cm to about 7.0 mS / cm, about 2.0 mS / cm to about 8.5 mS / cm, about 2.0 mS / cm to about 7.5 mS / cm, or about 2.5 mS / cm to about 7.0 mS / cm. In some embodiments, the conductivity for loading buffers and / or chase buffers of a target protein having a medium to high pI is about 2.5 mS / cm to about 7.0 mS / cm.

[0102] In some embodiments, the conductivity for loading buffers and / or chase buffers of a target protein having a medium to high pI is approximately 0.1 mS / cm, approximately 0.5 mS / cm, approximately 1.0 mS / cm, approximately 1.5 mS / cm, approximately 2.0 mS / cm, approximately 2.5 mS / cm, approximately 3.0 mS / cm, approximately 3.5 mS / cm, approximately 4.0 mS / cm, approximately 4.5 mS / cm, approximately 5.0 mS / cm, approximately 5.5 mS / cm, approximately 6.0 mS / cm, approximately 6.5 mS / cm, approximately 7.0 mS / cm, approximately 7.5 mS / cm, approximately 8.0 mS / cm, approximately 8.5 mS / cm, or approximately 9.0 mS / cm.

[0103] In some embodiments, the loading buffer and / or chase buffer for a target protein having a medium to high pI has a pH of about 7.0 to about 8.0 and a conductivity of about 2.0 mS / cm to about 7.0 mS / cm, for example, about 2.0 mS / cm, about 2.5 mS / cm, about 2.7 mS / cm, about 3.0 mS / cm, about 3.2 mS / cm, about 3.5 mS / cm, about 4.0 mS / cm, about 4.5 mS / cm, about 5.0 mS / cm, about 5.5 mS / cm, about 6.0 mS / cm, about 6.5 mS / cm, about 6.8 mS / cm, or about 7.0 mS / cm.

[0104] In some embodiments, the conductivity for loading buffers and / or chase buffers of a target protein having a low pI is approximately 6.0 mS / cm to 15.0 mS / cm, approximately 6.5 mS / cm to 15 mS / cm, approximately 7.0 mS / cm to 14.0 mS / cm, approximately 8.0 mS / cm to 13.0 mS / cm, approximately 8.0 mS / cm to 12.0 mS / cm, or approximately 9.0 mS / cm to 12.0 mS / cm.

[0105] In some embodiments, the conductivity for loading buffers and / or chase buffers of target proteins having low pI is approximately 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, 7.1 mS / cm, 7.2 mS / cm, 7.3 mS / cm, 7.4 mS / cm, 7.5 mS / cm, 7.6 mS / cm, 7.7 mS / cm, and 7.8 mS / cm. mS / cm, approximately 7.9mS / cm, approximately 8.0mS / cm, approximately 8.1mS / cm, approximately 8.2mS / cm, approximately 8.3mS / cm, approximately 8.4mS / cm, approximately 8.5mS / cm, approximately 8.6mS / cm, approximately 8.7mS / cm, approximately 8.8mS / cm, approximately 8.9mS / cm, approximately 9.0mS / c m, approximately 9.1mS / cm, approximately 9.2mS / cm, approximately 9.3mS / cm, approximately 9.4mS / cm, approximately 9.5mS / cm, approximately 9.6mS / cm, approximately 9.7mS / cm, approximately 9.8mS / cm, approximately 9.9mS / cm, approximately 10.0mS / cm, approximately 10.1mS / cm, approximately 10.2mS / cm, 10.3mS / cm, 10.4mS / cm, 10.5mS / cm, 10.6mS / cm, 10.7mS / cm, 10.8mS / cm, 10.9mS / cm, 11.0mS / cm, 11.1mS / cm, 11.2mS / cm, 11.3mS / cm, 11 .4mS / cm, approximately 11.5mS / cm, approximately 11.6mS / cm, approximately 11.7mS / cm, approximately 11.8mS / cm, approximately 11.9mS / cm, approximately 12.0mS / cm, approximately 12.1mS / cm, approximately 12.2mS / cm, approximately 12.3mS / cm, approximately 12.4mS / cm, approximately 12.5mS / cm, approximately 12.6mS / cm, approximately 12.7mS / cm, approximately 12.8mS / cm, approximately 12.9mS / cm, approximately 13.0mS / cm, approximately 13.1mS / cm, approximately 13.2mS / cm, approximately 13.3mS / cm, approximately 13.4mS / cm, approximately 13.5mS / cm, approximately 13.6mS / cm, 13.7mS / cm, 13.8mS / cm, 13.9mS / cm, 14.0mS / cm, 14.1mS / cm, 14.2mS / cm, 14.3mS / cm, 14.4mS / cm, 14.5mS / cm, 14.6mS / cm, 14.7mS / cm, 14.This corresponds to 8 mS / cm, approximately 14.9 mS / cm, or approximately 15.0 mS / cm.

[0106] In some embodiments, the loading buffer and / or chase buffer for the target protein having a low pI has a pH of about 7.0 to about 8.0 and a conductivity of about 8.0 mS / cm to 13.0 mS / cm.

[0107] In some embodiments, the target protein has a pI of about 7.0 to about 8.0, for example, about 7.6, and the loading buffer and chase buffer for the protein have the same pH of about 7.0 to about 8.0, for example, about 7.6, and conductivity of 3.0 mS / cm to 6.0 mS / cm, for example, 3.2 mS / cm and 5.5 mS / cm, respectively.

[0108] In another embodiment, the target protein has a pI of about 8.0 to about 9.0, for example, about 8.4, and the loading buffer and chase buffer for the protein have the same pH of about 8.0 to about 9.0, for example, about 8.4, and conductivity of 2.0 mS / cm to 4.0 mS / cm, for example, 2.7 mS / cm and 3.0 mS / cm, respectively.

[0109] In some embodiments, the target protein has a pI of 8.5–9.5, for example, about 8.9, and the loading buffer and chase buffer for the protein have the same pH of 8.5–9.5, for example, about 8.9, and conductivity of 2.0 mS / cm–5.0 mS / cm, for example, 3.0 mS / cm and 4.5 mS / cm, respectively.

[0110] In another embodiment, the target protein has a pI of about 9.0 to about 10.0, for example, about 9.2, and the loading buffer and chase buffer for the protein have the same pH of about 9.0 to about 10.0, for example, about 9.2, and conductivity of 6.0 mS / cm to 7.0 mS / cm, for example, 6.8 mS / cm and 6.8 mS / cm, respectively.

[0111] In another embodiment, the target protein has a pI of about 5.0 to about 6.0, for example, about 5.8, the loading buffer and chase buffer for the protein have the same pH of about 5.0 to about 6.0, for example, about 5.8, and conductivity of 9.0 mS / cm to 12.0 mS / cm, for example, 9.2 mS / cm and 12.0 mS / cm, respectively.

[0112] The methods described herein (II and III) may be used to purify proteins in a mixture containing impurities including host cell proteins (HCP), DNA, high molecular weight proteins (HMW), low molecular weight proteins (LMW), residual protein A (rPA), or any combination thereof. In some embodiments, chromatography reduces (i) DNA to a level of about 20 pg / mL or less, (ii) LMW to a level of about 1.0% or less, (iii) HMWs to a level of about 1.0% or less, (iv) HCP to a level of about 100 ppm or less, (v) residual protein A (rPA) to a level of about 6 ppm or less, or any combination thereof.

[0113] In some embodiments, chromatography reduces the DNA to levels of approximately 20 pg / mL or less, approximately 18 pg / mL or less, approximately 16 pg / mL or less, approximately 14 pg / mL or less, approximately 12 pg / mL or less, approximately 10 pg / mL or less, approximately 8 pg / mL or less, approximately 6 pg / mL or less, approximately 4 pg / mL or less, or approximately 2 pg / mL or less.

[0114] In some embodiments, chromatography reduces the low molecular weight (LMW) to levels of approximately 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

[0115] In some embodiments, chromatography reduces the high molecular weight (HMW) to levels of approximately 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%.

[0116] In some embodiments, chromatography reduces host cell proteins (HCPs) to levels of approximately 100 ppm or less, approximately 90 ppm or less, approximately 80 ppm or less, approximately 70 ppm or less, approximately 60 ppm or less, approximately 50 ppm or less, approximately 40 ppm or less, approximately 30 ppm or less, approximately 20 ppm or less, or approximately 10 ppm or less.

[0117] In some embodiments, chromatography reduces the residual protein A (rPA) to levels of approximately 6 ppm or less, approximately 5 ppm or less, approximately 4 ppm or less, approximately 3 ppm or less, approximately 2 ppm or less, approximately 1 ppm or less, approximately 0.8 ppm or less, approximately 0.6 ppm or less, approximately 0.5 ppm or less, approximately 0.4 ppm or less, approximately 0.3 ppm or less, approximately 0.2 ppm or less, or approximately 0.1 ppm or less.

[0118] In some embodiments, the method of the Disclosure is carried out after one or more downstream purification steps, such as filtration, affinity chromatography, tangential flow chromatography, virus inactivation, or any combination thereof. In other embodiments, the method of the Disclosure is carried out without a post-collection purification step. In other embodiments, the chromatography of the Disclosure is the final step before virus inactivation. In yet another embodiment, the method of the Disclosure inactivates one or more viruses simultaneously. In yet another embodiment, the method of the Disclosure further includes virus inactivation before or after AEX and CEX or a combination of CEX and AEX.

[0119] III. Method for removing buffer transition peaks This disclosure also includes methods for reducing or removing buffer migration peaks between a loading buffer and a chase buffer. To reduce or remove buffer migration peaks in optionally flow-through mode chromatography, such as AEX, CEX, a combination of AEX and CEX, or a combination of CEX and AEX, the method includes adding a loading buffer and a chase buffer, wherein the chase buffer has a higher conductivity than the loading buffer. In some embodiments, the chromatography is a combination of AEX and CEX or a combination of CEX and AEX.

[0120] In some embodiments, the conductivity of the chase buffer is at least 0.5 mS / cm, at least 0.6 mS / cm, at least 0.7 mS / cm, at least 0.8 mS / cm, at least 0.9 mS / cm, at least 1.0 mS / cm, at least 1.1 mS / cm, at least 1.2 mS / cm, at least 1.3 mS / cm, at least 1.4 mS / cm, at least 1.5 mS / cm, at least 1.6 mS / cm, at least 1.7 mS / cm, at least 1.8 mS / cm, at least 1.9 mS / cm, at least 2.0 mS / cm, at least 2.1 mS / cm, at least 2.2 mS / cm, at least 2.3 mS / cm, at least 2.4 mS / cm, at least 2.5 mS / cm, and at least 2. 6 mS / cm, at least 2.7 mS / cm, at least 2.8 mS / cm, at least 2.9 mS / cm, at least 3.0 mS / cm, at least 3.1 mS / cm, at least 3.2 mS / cm, at least 3.3 mS / cm, at least 3.4 mS / cm, at least 3.5 mS / cm, at least 3.6 mS / cm, at least 3.7 mS / cm, at least 3.8 mS / cm, at least 3.9 mS / cm, at least 4.0 mS / cm, at least 4.1 mS / cm, at least 4.2 mS / cm, at least 4.3 mS / cm, at least 4.4 mS / cm, at least 4.5 mS / cm, at least 4.6 mS / cm, at least 4.7 mS / cm, at least 4.8 mS / cm, at least 4.9 mS / cm, or at least 5.0 mS / cm higher. In another embodiment, the conductivity of the loading buffer is approximately 1.0 mS / cm higher than that of the chase buffer.

[0121] In some embodiments, the loading buffer has a lower conductivity than the chase buffer. In some embodiments, the conductivity of the loading buffer is at least 0.5 mS / cm, at least 1.0 mS / cm, at least 1.5 mS / cm, at least 2.0 mS / cm, at least 2.5 mS / cm, at least 3.0 mS / cm, at least 3.5 mS / cm, at least 4.0 mS / cm, at least 4.5 mS / cm, or at least 5.0 mS / cm lower than that of the chase buffer. In other embodiments, the conductivity of the loading buffer is about 1.0 mS / cm lower than that of the chase buffer.

[0122] In some embodiments, the method for reducing or removing the buffer migration peak of (III) may be combined with the method for purifying the protein of (II). Accordingly, in some embodiments, the Disclosure includes a method for purifying a target protein, the method comprising performing AEX-CEX combined or CEX-AEX combined chromatography under operating conditions (e.g., pH and / or conductivity) in which automation, engineering control and / or in-line adjustment is not used between AEX and CEX, and the chase buffer used in chromatography has a higher conductivity than the loading buffer used in chromatography.

[0123] Target protein Methods (II) and / or (III) of this disclosure may be used to isolate or purify a target protein, e.g., a natural or non-natural protein, a recombinant or plasma-purified protein, or any combination thereof. The target protein may be a mixture with one or more contaminants as described in any part of this specification. In some embodiments, the target protein has already undergone downstream purification, e.g., filtration, affinity chromatography, tangential flow chromatography, virus inactivation, polishing, or any combination thereof. In other embodiments, the target protein is a mixture with contaminants and has not undergone post-isolation purification.

[0124] In some embodiments, the mixture contains the target protein, which is an antibody.

[0125] In some embodiments, the target protein is a fusion protein containing the target protein and a heterologous moiety. In other embodiments, the fusion protein isolated or purified by this method is an Fc fusion protein. In other embodiments, the protein isolated or purified by this method is a pegylated, hesylated, or sialylated protein.

[0126] In other embodiments, the target protein is produced in host cells. In some embodiments, the target protein is produced in a culture containing mammalian cells. In some embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, HEK293 cells, mouse myeloma (NS0), baby hamster kidney cells (BHK), monkey kidney fibroblasts (COS-7), Madin-Derby bovine kidney cells (MDBK), or any combination thereof. In some embodiments, the starting mixture may be harvested cell culture medium, cell culture supernatant, conditioned cell culture supernatant, cell lysate, and clarified bulk.

[0127] In some embodiments, the cell culture is subjected to one or more downstream purification methods, such as affinity chromatography selected from protein A affinity chromatography and protein G affinity chromatography. In other embodiments, the starting mixture of the method undergoes one or more downstream purifications, such as diafiltration, ultrafiltration, affinity chromatography, tangential flow chromatography, virus inactivation, or any combination thereof.

[0128] This disclosure is further illustrated by the following embodiments, which should not be construed as further limitations. The contents of all references cited throughout this application are expressly incorporated herein by attribution. [Examples]

[0129] Example 1: Evaluation of polishing To evaluate the effectiveness of polishing process sequences and combinations of polishing processes, as well as the reduction of unwanted contaminants, various polishing plans were examined as shown in Table 1. The loading material for testing was a protein A elution and neutralization pool (PAVIN) at pH 5.5 (without depth filtration). Before each run, the PAVIN pool was adjusted to pH 7.2 using 2M Tris. As shown in Table 1, CEX(F / T) effectively removed HMW and rPA; AEX(F / T) significantly reduced HCP but could not reduce rPA and MW. Using a two-step polishing (AEX(F / T) after CEX(F / T)), HCP, HMW, and rPA levels were all reduced to relatively low levels. Here, the CEX-AEX F / T combination was also evaluated by connecting two columns and operating under CEX F / T conditions (loading at pH 7.2 and 5 mS / cm, tracking at pH 7.2 and 6 mS / cm). As shown in Figure 1, the outlet of a POROS XS column (bed height 10 cm, inner diameter 0.66 cm) was directly connected to the inlet of a Capto Q column (bed height 10 cm, inner diameter 0.66 cm) without automation or engineering control between the two columns.

[0130] To evaluate the effectiveness of the unadjusted CEX / AEX F / T (AEX / CEX F / T) combination polishing process, the starting mixture "Protein A Elution and Neutralization Pool" (PAVIN) had a pH of 5.5 and contained the impurities listed in Table 1. The reduction of unwanted contaminants after various chromatographic approaches is shown in detail in Table 1. CEX alone, AEX alone, the conductivity-adjusted CEX-AEX combination, and the unadjusted CEX-AEX followed by the AEX-CEX combination were tested under the operating conditions described above. All chromatography was performed in flow-through mode.

[0131] The unadjusted CEX-AEX or AEX-CEXF / T combined polishing process demonstrated significant removal of HCP, rPA, and HMW, while reducing operation time / cost and skid footprint. Numerous advantages of CEX-AEX or AEX-CEXF / T combined polishing were demonstrated; these include significant reductions in operation time, cost, and skid footprint compared to a single F / T polishing unit and a CEX(F / T) and AEX(F / T) combined polishing process with conductivity adjustment between CEX and AEX. The CEX-AEX or AEX-CEX combination without pH and conductivity adjustment showed significantly improved downstream productivity compared to a single F / T polishing unit. Both CEX and AEX(F / T) operate at high loads (300 g / L or higher). The CEX and AEX or AEX and CEX chromatography combination without pH and conductivity adjustment can utilize the same buffer system where CEX and AEX have the same charge, EQ, strip, purification, and storage buffers. Current chromatography can also be implemented simply by directly connecting the outlet of the CEX to the inlet of the AEX, as shown in Figure 1. [Table 1]

[0132] Example 2: Overview of Molecules Evaluated by CEX-AEX F / T Polishing The effectiveness of purification was measured for numerous antibodies with varying isoelectric points (pI), and details are shown in Table 2. The first four antibodies were purified using a pH lower than their isoelectric point, indicating that the antibodies carried a slightly positive net charge during chromatography. To prevent antibody binding to the column and facilitate passage, the conductivity of the buffer was increased using salts to a level where the target antibody was not strongly retained on the column, but unwanted contaminants such as HCP, rPA, and HMW were retained. For the fifth antibody with a pI of 5.8, a higher salt concentration was used to prevent interaction between the negatively charged antibody and the anion exchange resin. [Table 2]

[0133] A typical chromatogram of the CEX-AEXF / T combined polishing process without adjustment is shown in Figure 2.

[0134] Example 3 Evaluation of the peak after buffer transfer-induced loading The effectiveness of CEX(F / T) in separating antibody-3 was evaluated by passing a protein mixture containing antibody-3 through CEX(F / T). CEX F / T was performed at pH 7.2 using a 5 mS / cm loading buffer and a 5 mS / cm chase buffer. Both buffers contained sodium acetate-Tris buffer. Protein elution was measured by absorbance at 280 nm. As shown in Figure 3A, the transfer from the loading buffer to the chase buffer induced an unwanted post-buffer transfer peak. When CEX F / T was performed again with only the conductivity of the chase buffer changed, i.e., increased by 1 mS / cm to 6 mS / cm, the chromatogram showed the disappearance of the post-buffer transfer peak, as shown in Figure 3B. Therefore, increasing the conductivity of the chase buffer can reduce or eliminate the peak caused by the transfer from the loading buffer to the chase buffer.

[0135] Throughout this application, various publications are referenced in parentheses by author name and date, or by patent number or patent publication number. The disclosures in these publications are incorporated as a whole by attribution to better describe the state of the art known to those skilled in the art as of the date of the disclosure described and claimed herein. However, the references to references herein should not be construed as an acknowledgment that such references constitute prior art to this disclosure.

Claims

1. A method for purifying an antibody from a mixture containing one or more contaminants, comprising performing a combination of cation exchange chromatography (CEX) and anion exchange chromatography (AEX) or a combination of AEX and CEX ("combined chromatography") in flow-through mode under specific operating conditions, wherein the specific operating conditions are that the AEX and CEX are directly connected, no automation, engineering control, or in-line adjustment is used between the AEX and CEX, and the pH and conductivity between the AEX and CEX are not adjusted, and the mixture contains Protein A before the combined chromatography. A method comprising: isolation by affinity chromatography, wherein the combination chromatography comprises adding a loading buffer and adding a chase buffer, wherein the pH of the chase buffer is the same as the pH of the loading buffer, the conductivity of the chase buffer is higher than the conductivity of the loading buffer, the impurities comprise host cell proteins (HCP), DNA, high molecular weight proteins (HMW), low molecular weight proteins (LMW), residual protein A (rPA), or any combination thereof, and the combination chromatography reduces the rPA level to 0.6 ppm or less.

2. The method according to claim 1, wherein the antibody has an isoelectric point (pI) higher than 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

0.

3. The method according to claim 2, wherein the antibody has a pI lower than 12.0, 11.9, 11.8, 11.7, 11.6, 11.5, 11.4, 11.3, 11.2, 11.1, 11.0, 10.9, 10.8, 10.7, 10.6, 10.5, 10.4, 10.3, 10.2, 10.1, or 10.

0.

4. The method according to claim 1, wherein the antibody has a pI of 7.0-11.0, 7.0-10.0, 6.5-10.5, or 7.2-9.

6.

5. The method according to claim 1, wherein the antibody has a pI of 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, or 11.

0.

6. The method according to claim 1, wherein the antibody has a pI lower than 6.5, 6.4, 6.3, 6.2, 6.1, 5.9, 5.8, or 5.

7.

7. The method according to claim 6, wherein the antibody has a pI higher than 1.0, 2.0, 3.0, 4.0, or 5.

0.

8. The method according to claim 1, wherein the antibody has a pI of 1.0 to 6.5, 2.0 to 6.0, 2.5 to 6.5, or 2.0 to 5.

8.

9. The method according to claim 1, wherein the antibody has a pI of 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, or 5.

5.

10. The method according to any one of claims 1 to 9, wherein the loading buffer has a pH higher than 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.

0.

11. The method according to any one of claims 1 to 9, wherein the loading buffer has a pH of 6.0 to 11.0, 6.5 to 11.0, 7.0 to 10.5, 7.0 to 10.0, 7.0 to 9.5, 7.0 to 9.0, 7.0 to 8.5, 7.0 to 8.0, or 7.0 to 7.

5.

12. The method according to any one of claims 1 to 9, wherein the loading buffer has a pH of 6.5, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4, 9.6, 9.8, 10.0, 10.5, 11.0, or 11.

5.

13. The method according to any one of claims 1 to 10, wherein the chase buffer has a pH higher than 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.

0.

14. The method according to any one of claims 1 to 10, wherein the chase buffer has a pH of 6.0 to 11.0, 6.5 to 10.0, 7.0 to 11.0, 7.0 to 10.0, 7.0 to 9.0, 7.0 to 8.0, 7.0 to 8.5, 7.0 to 9.5, 6.5 to 8.5, or 6.5 to 7.

5.

15. The method according to any one of claims 1 to 10, wherein the chase buffer has a pH of 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, or 9.

0.

16. The method according to any one of claims 1 to 10, wherein the loading buffer has a conductivity of 0.1 mS / cm to 9.0 mS / cm, 0.5 mS / cm to 9.0 mS / cm, 1.0 to 9.0 mS / cm, 2.0 to 8.0 mS / cm, 2.0 to 7.0 mS / cm, 2.0 to 8.5 mS / cm, 2.0 to 7.5 mS / cm, or 2.5 to 7.0 mS / cm.

17. The method according to claim 16, wherein the loading buffer has a conductivity of 2.5 mS / cm to 7.0 mS / cm.

18. The method according to claim 16, wherein the loading buffer has a conductivity of 0.1 mS / cm, 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4.0 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 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, or 9.0 mS / cm.

19. The method according to any one of claims 1 to 10, wherein the loading buffer has a conductivity of 6.0 mS / cm to 15.0 mS / cm, 6.5 mS / cm to 15 mS / cm, 7.0 mS / cm to 14.0 mS / cm, 8.0 mS / cm to 13.0 mS / cm, or 9.0 mS / cm to 12.0 mS / cm.

20. The method according to claim 19, wherein the loading buffer has a conductivity of 9.0 to 9.5 mS / cm.

21. The method according to claim 19, wherein the loading buffer has a conductivity of 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, 10.0 mS / cm, 10.5 mS / cm, 11.0 mS / cm, 11.5 mS / cm, 12.0 mS / cm, 12.5 mS / cm, 13.0 mS / cm, 13.5 mS / cm, 14.0 mS / cm, 14.5 mS / cm, or 15.0 mS / cm.

22. The method according to any one of claims 1 to 10, wherein the chase buffer has a conductivity of 0.1 mS / cm to 9.0 mS / cm, 0.5 mS / cm to 9.0 mS / cm, 1.0 to 9.0 mS / cm, 2.0 to 8.0 mS / cm, 2.0 to 7.0 mS / cm, 2.0 to 8.5 mS / cm, 2.0 to 7.5 mS / cm, or 2.5 to 7.0 mS / cm.

23. The method according to claim 22, wherein the chase buffer has a conductivity of 0.1 mS / cm, 0.5 mS / cm, 1.0 mS / cm, 1.5 mS / cm, 2.0 mS / cm, 2.5 mS / cm, 3.0 mS / cm, 3.5 mS / cm, 4.0 mS / cm, 4.5 mS / cm, 5.0 mS / cm, 5.5 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, or 9.0 mS / cm.

24. The method according to any one of claims 1 to 10, wherein the chase buffer has a conductivity of 6.0 mS / cm to 15.0 mS / cm, 6.5 mS / cm to 15.0 mS / cm, 7.0 to 15.0 mS / cm, 8.0 to 14.0 mS / cm, 8.0 to 13.0 mS / cm, 8.0 to 14.5 mS / cm, 8.0 to 13.5 mS / cm, or 8.5 to 13.0 mS / cm.

25. The method according to claim 24, wherein the chase buffer has a conductivity of 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, 10.0 mS / cm, 10.5 mS / cm, 11.0 mS / cm, 11.5 mS / cm, 12.0 mS / cm, 12.5 mS / cm, 13.0 mS / cm, 13.5 mS / cm, 14.0 mS / cm, 14.5 mS / cm, or 15.0 mS / cm.

26. The method according to claim 22 or 24, wherein the conductivity of the chase buffer is at least 0.1 mS / cm, at least 0.2 mS / cm, at least 0.3 mS / cm, at least 0.4 mS / cm, at least 0.5 mS / cm, at least 1.0 mS / cm, at least 1.5 mS / cm, at least 2.0 mS / cm, at least 2.5 mS / cm, at least 3.0 mS / cm, at least 3.5 mS / cm, at least 4.0 mS / cm, at least 4.5 mS / cm, or at least 5.0 mS / cm higher than the conductivity of the loading buffer.

27. The method according to any one of claims 1 to 10, wherein combination chromatography reduces the HCP level to 100 ppm or less, 90 ppm or less, 80 ppm or less, 70 ppm or less, 60 ppm or less, 50 ppm or less, 40 ppm or less, 30 ppm or less, 20 ppm or less, or 10 ppm or less.

28. The method according to any one of claims 1 to 10, wherein combination chromatography reduces the HMW level to 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

29. The method according to any one of claims 1 to 10, wherein combination chromatography reduces the LMW level to 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less.

30. The method according to any one of claims 1 to 10, wherein combination chromatography reduces the DNA level to 20 pg / mL or less, 18 pg / mL or less, 16 pg / mL or less, 14 pg / mL or less, 12 pg / mL or less, 10 pg / mL or less, 8 pg / mL or less, 6 pg / mL or less, 4 pg / mL or less, or 2 pg / mL or less.

31. The method according to any one of claims 1 to 10, wherein combination chromatography reduces the rPA level to 0.5 ppm or less, 0.4 ppm or less, 0.3 ppm or less, 0.2 ppm or less, or 0.1 ppm or less.

32. The method according to any one of claims 1 to 10, wherein the loading buffer comprises sodium chloride, ammonium chloride, potassium chloride, sodium acetate, ammonium acetate, sodium sulfate, ammonium sulfate, ammonium thiocyanate, sodium citrate, sodium phosphate, and their potassium salts, magnesium salts, and calcium salts, or any combination thereof.

33. The method according to claim 32, wherein the loading buffer comprises sodium acetate-Tris.

34. The method according to any one of claims 1 to 10, wherein the chase buffer comprises sodium chloride, ammonium chloride, potassium chloride, sodium acetate, ammonium acetate, sodium sulfate, ammonium sulfate, ammonium thiocyanate, sodium citrate, sodium phosphate, and their potassium salts, magnesium salts, and calcium salts, or any combination thereof.

35. The method according to claim 34, wherein the chase buffer comprises sodium acetate-Tris.

36. The method according to any one of claims 1 to 35, wherein the chromatography includes a column loaded with 50 g / L resin, 100 g / L resin, 150 g / L resin, 200 g / L resin, 250 g / L resin, 300 g / L resin, 350 g / L resin, 400 g / L resin, 450 g / L resin, or 500 g / L resin.

37. Chromatography is Poros （商標） HS, Poros （商標） XS, carboxymethyl-cellulose, BAKERBOND （登録商標） ABX （商標） , sulfopropyl immobilized on agarose and sulfonyl immobilized on agarose, MonoS （登録商標） , MiniS （登録商標） , Source （商標） 15S, 30S, SP SEPHAROSE （商標） , CM SEPHAROSE （商標） BAKERBOND （登録商標） Carboxy-Sulfo, WP CBX, WP Sulfonic, Hydrocell （商標） , CM, Hydrocel （商標） , SP, UNOSphere （商標） , S, Macro-Prep （登録商標） , High S, Macro-Prep （登録商標） , CM, Ceramic HyperD （登録商標） [[ID=三十二]] （登録商標） , CM, Ceramic HyperD （登録商標） , Z, Trisacryl （登録商標） , M CM, Trisacryl （登録商標） , LS CM, Trisacryl （登録商標） , M SP, Trisacryl （登録商標） , LS SP, Sephadex （登録商標） , LS SP, DOWEX （商標） , Fine Mesh Strong Acid Cation Resin, DOWEX （商標） , MAC-3, Matrix （登録商標） , Cellufine （商標） , C500, Matrix （登録商標） , Cellufine （商標） , C200, Fractogel （登録商標） , EMD SO3-, Fractogel （登録商標） , EMD SE, Fractogel （登録商標） , EMD COO-, Amberlite （商標） ​Weak and Strong Cation Exchangers, Diaion （商標） Weak and Strong Cation Exchangers, TSK Gel （登録商標） SP-5PW-HR, TSK Gel （登録商標） SP-5PW, Toyopear （登録商標） l CM (650S, 650M, 650C), Toyopearl （登録商標） SP (650S, 650M, 650C), CM (23, 32, 52), SE (52, 53), P11, Express （登録商標） - Ion C, and Express （登録商標） The method according to claim 36, comprising a CEX resin selected from -Ion S, or any combination thereof.

38. クロマカラフーが、POROS （商標） HQ、POROS （商標） XQ、Q SEPHAROSE （商標） Fast Flow、BEAE SEPHAROSE （商標） Fast low、ARTOBIND （登録商標） Q、ANX SEEPHAROSE （商標） 4 Fast Flow (･･･ sub),Q SEPHAROSE （商標） ^L、Q. SEEPHAROSE （商標） big beads, DEAE @ （登録商標） A-25、Date of birth （登録商標） A-50, QA （登録商標） A-25、QA-E Sepladex （登録商標） A-50、Q SEPHAROSE （商標） highh piformmanceQ （商標） XL、Source （商標） 15Q、Source （商標） 30Q、Resource （商標） Q、Captain （商標） Q、Captain （商標） DEA、MONO （登録商標） Q. （登録商標） Super QS （登録商標） DEA, TOYO PEARL （登録商標） QAE、Toyopeerl （登録商標） Q. （登録商標） GOLCLQLLLLLLL （登録商標） SuperQ、TS gel （登録商標） DEA, FRACTURE （登録商標） EMD TMAE、F･････ （登録商標） EMD TMAE HCCU、F｡actｯse （登録商標） EMD DEAE、Aractogel （登録商標） EMD DMAE、MAocrp （登録商標） High Q、Mro-rrp （登録商標） -DEA, Unosphere （商標） Q, Nuvia （商標） Q, POROS （商標） PI, DEAE Ceramic HyperD （登録商標） ,Q Ceramic HyperD （登録商標） The method according to claim 36, comprising an AEX resin selected from, or any combination thereof.

39. The method according to any one of claims 1 to 10, wherein the antibody is an isotype selected from IgM, IgA, IgE, IgD, and IgG.

40. The method according to claim 39, wherein the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4.

41. The method according to any one of claims 1 to 10, wherein chromatography inactivates the virus.

42. The method according to any one of claims 1 to 10, further comprising inactivating a virus.

43. The method according to claim 1, wherein the starting mixture for protein A affinity chromatography is selected from collected cell culture medium, cell culture supernatant, conditioned cell culture supernatant, cell lysate, and clarified bulk.

44. The method according to claim 43, wherein the starting mixture is derived from mammalian cell culture.

45. The method according to claim 44, wherein the mammalian cells are Chinese hamster ovary (CHO) cells.

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