Methods of purifying proteins 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 industry. Generally, production and purification are first performed by various "upstream" cell culture processes to produce the protein of interest. After production, "downstream" purification processes are used to separate and isolate the protein of interest from any undesirable contaminants that may also be present in the mixture produced in the upstream process. Various upstream and downstream technologies can be found for example in Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes, Jagschies et al., 2017. A particular subset of proteins, monoclonal antibodies (mAbs) and their derivative products (e.g. Fc fusion proteins, bispecific antibodies), play a key role in the treatment of the most challenging human diseases due to the safety, efficacy and high quality of these types of biologics. The production of mAbs starts with protein expression in recombinant mammalian cultures. The clarified bulk is then subjected to downstream purification (DSP). Downstream purification processes typically use Protein A chromatography for capture followed by one or two polishing steps 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. Cation exchange (CEX), anion exchange (AEX), hydrophobic interaction (HIC) and mixed chromatography have all been used as polishing steps.

[0002] Current downstream purification is often performed as a batch process with hold tanks between different unit operations (chromatography and filters). Necessary buffer adjustments (e.g., pH or conductivity adjustments) are often required for each unit operation. Automation and in-line adjustment of eluents have been reported to improve purification steps. Nevertheless, in-line adjustment of buffer conditions can be cumbersome and increase operational costs and time as well as skid footprints. Summary of the Invention

[0003] The present disclosure relates to a combination chromatography process for continuous purification of a protein of interest, in which AEX and CEX are operated in a flow-through mode without in-line adjustment of the chromatographic conditions, including pH and / or conductivity. One aspect of the present disclosure relates to the purification of a protein of interest using an AEX-CEX or CEX-AEX purification approach, without in-line adjustment of any of the purification conditions, including pH or conductivity. Specifically, the approach uses tandem cation exchange chromatography (CEX) and anion exchange chromatography (CEX) in either AEX-CEX or CEX-AEX order. In some embodiments, AEX and CEX are not adjusted for pH or conductivity of the in-process pool between AEX and CEX or CEX and AEX. Another feature of the present disclosure is that the column conditions of AEX and CEX are processed together and operated as a single unit, which significantly reduces the process cost, process operation time and process skid footprint.

[0004] In some embodiments, the protein has an isoelectric point (pI) greater than about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0. In other embodiments, the protein has an isoelectric point (pI) less than about 12.0, about 11.9, about 11.8, about 11.7, about 11.6, about 11.5, about 11.4, about 11.3, about 11.2, about 11.1, about 11.0, about 10.9, about 10.8, about 10.8, about 10.7, about 10.6, about 10.5, about 10.4, about 10.3, about 10.2, about 10.1 or about 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) of about 1.0, about 2.0, about 3.0, about 4.0 or higher than 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, the combined chromatography comprises adding a loading buffer and adding a chase buffer.

[0008] In some embodiments, the loading buffer has a pH of about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or higher than about 10.0. In other embodiments, the loading buffer has a pH of about 6.0 to about 11.0, about 6.5 to about 11.0, about 7.0 to about 10.5, about 7.0 to about 10.0, about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 7.5. In other embodiments, the loading 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.

[0009] In some embodiments, the chase buffer has a pH greater than about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0. In other 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. In other embodiments, the chase buffer has a pH of about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, 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 or about 9.0.

[0010] In some embodiments, the chase buffer has a pH that is the same or different from the pH of the loading buffer.

[0011] In some embodiments, the loading buffer has a conductivity of 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 to about 9.0 mS / cm, about 2.0 to about 8.0 mS / cm, about 2.0 to about 7.0 mS / cm, about 2.0 to about 8.5 mS / cm, about 2.0 to about 7.5 mS / cm, or about 2.5 to about 7.0 mS / cm. In other embodiments, the loading buffer has a conductivity of about 0.1 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 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 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm or about 9.0 mS / cm.

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

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

[0014] In some embodiments, the conductivity of the chase buffer is higher, lower or the same as the conductivity 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 the conductivity of the loading buffer.

[0015] In some embodiments, the mixture comprises one or more contaminants. In some embodiments, the contaminants comprise host cell proteins (HCPs), DNA, high molecular weight species (HMW), low molecular weight species (LMW), residual protein A (rPA), or any combination thereof.

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

[0017] Also described is a method for reducing or removing a buffer migration peak after a loading step in chromatography, the method comprising adding a loading buffer and a chase buffer during chromatography for purifying a protein in a mixture, the chase buffer having 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 its 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 its 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 up to 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 using Poros HS, Poros XS, carboxy-methyl-cellulose, BAKERBOND ABX TM , sulfopropyl immobilized on agarose and sulfonyl immobilized on agarose, MonoS, MiniS, Source 15S, 30S, SP SEPHAROSE TM , C.M. 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 and a CEX resin 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, the chromatography is performed using POROS HQ, POROS XQ, 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, Resource 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 AEX resin selected from DEAE, Fractogel EMD DMAE, Macroprep High Q, Macro-prep-DEAE, Unosphere Q, Nuvia Q, PORGS PI, DEAE Ceramic HyperD, Q Ceramic HyperD, or any combination thereof.

[0020] In some embodiments, the protein is selected from an antibody or antibody fragment, a fusion protein, a natural 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, the 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 a 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 combined CEX-AEX or AEX-CEX polishing. The outlet of column 1 is directly connected to the outlet of column 2. No automation, engineering control or regulation exists or is made between these two columns.

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

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

[0025] [Figure 3B]Figure 3B shows the chromatogram of CEX F / T loaded at 5 mS / cm, pH 7.2 and chased at 6 mS / cm, pH 7.2. The use of a higher conductivity buffer during the chase effectively removes the post-loading peak. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present 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, AEX and CEX are not adjusted for buffer pH or conductivity between AEX and CEX or CEX and AEX. Another feature of the present disclosure is that the column conditions of AEX and CEX are processed together and done so as a single unit, which significantly reduces process cost, process operation time and process skid footprint.

[0027] As shown in the examples, the approach is effective in removing unwanted contaminants in a mixture of target proteins with a wide range of isoelectric points. By using such an AEX-CEX system, both CEX and AEX processes can be improved simultaneously. In certain embodiments, the present 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.), where CEX and AEX are operated in flow-through mode, and the target protein does not bind strongly to either CEX or AEX. By using such a system that uses CEX and AEX to remove unwanted contaminants from a mixture, extremely high throughput is possible.

[0028] In certain embodiments, the present disclosure provides a method for purifying a protein of interest from a mixture that contains the protein of interest and one or more contaminants.Possible contaminants include host cell proteins (HCPs), high molecular weight species (HMW), low molecular weight species (LMW), DNA, and / or residual protein A (rPA) from a previous protein A capture step.The present disclosure also provides a method for reducing buffer transition post-loading peaks, in which the chromatography process is interrupted when the loading step transitions to a post-loading chase buffer for chasing the mixture off the column.

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

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

[0031] Whenever an embodiment is described herein with the term "comprising," it is understood that other similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of."

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.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 those skilled in the art with a general dictionary of many terms used in this disclosure.

[0033] Units, prefixes and symbols are shown in the format accepted by the International System of Units (SI). Numerical ranges include the numerical values ​​that define the range. The headings provided herein are not limitations of the various aspects of the disclosure, but may be had by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

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

[0035] The term "about" or "essentially includes" refers to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measurement system. For example, "about" or "essentially includes" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "essentially includes" can mean within a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean values ​​up to an order of magnitude or up to 5-fold. When a particular value or composition is provided in the present application and claims, unless otherwise indicated, the meaning of "about" or "essentially includes" should be assumed to be within an acceptable error range of the particular value or composition.

[0036] Any concentration range, percentage range, ratio range, or integer range described herein should be understood to include every integer value within the described range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers), unless otherwise stated.

[0037] As used herein, the term "protein of interest" is used in its broadest sense to include any protein (either natural or recombinant) present in a mixture from which purification is desired. Proteins of interest include, but are not limited to, enzymes, hormones, growth factors, cytokines, immunoglobulins (e.g., antibodies), and / or any fusion protein.

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

[0039] The term "virus inactivation" refers to the process of removing infectious virus contaminants from a mixture. Currently, there are many different methods for inactivating infectious pathogenic viruses, including, for example, heat inactivation, organic solvent / detergent (S / D) inactivation, pH inactivation, chemical inactivation, and / or ultraviolet radiation inactivation.

[0040] The term "chromatography" refers to any type of technique that separates a protein of interest (e.g., an antibody) from other molecules (e.g., contaminants) present in a mixture. Typically, the protein of interest is separated from other molecules (e.g., contaminants) as a result of differences in the rate at which individual molecules of the mixture move through a stationary medium under the influence of a mobile phase, or as a result of binding and elution processes. The terms "matrix" or "chromatography matrix" are used interchangeably herein and refer to any type of adsorbent, resin, or solid phase that separates the protein of interest (e.g., a protein containing an Fc region, such as an immunoglobulin) from other molecules present in the mixture in a separation process. Non-limiting examples include particulate, monolithic, or fibrous resins, as well as membranes that may be placed in columns or cartridges. Examples of materials that form matrices include polysaccharides (e.g., agarose and cellulose); other mechanically stable matrices such as silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above. Examples of typical matrix types suitable for the method of the present disclosure are cation exchange resin, affinity resin, anion exchange resin or mixed resin. A "ligand" is a functional group that binds to a chromatography matrix and determines the binding properties of the matrix. Examples of "ligands" include, but are not limited to, ion exchange groups, hydrophobic interaction groups, hydrophilic interaction groups, thiophilic interaction groups, metal affinity groups, affinity groups, bioaffinity groups and mixed groups (combinations of the above). Some preferred ligands that can be used herein include, but are not limited to, strong cation exchange groups, such as sulfopropyl, sulfonic acid; strong anion exchange groups, such as trimethylammonium chloride; weak cation exchange groups, such as carboxylic acid; weak anion exchange groups, such as N5N diethylamino or DEAE; hydrophobic interaction groups, such as phenyl, butyl, propyl, hexyl; affinity groups, such as protein A, protein G, protein L. In order to make the present disclosure more easily understandable, certain terms are first defined.As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0041] The term "affinity chromatography" refers to a protein separation technique in which a protein of interest (e.g., a protein of interest or an antibody containing an Fc region) specifically binds to a ligand specific for the protein of interest. Such ligands are commonly referred to as biospecific ligands. In some embodiments, the biospecific ligand (e.g., protein A or a functional variant thereof) is covalently bound to a chromatography matrix material and is accessible to the protein of interest in solution as the solution contacts the chromatography matrix. The protein of interest generally retains its specific binding affinity for the biospecific ligand during the chromatography step, while other solutes and / or proteins in the mixture do not appreciably or specifically bind to the ligand. Binding of the protein of interest to the immobilized ligand allows contaminating proteins or protein impurities to pass through the chromatography matrix while the protein of interest remains specifically bound to the immobilized ligand on the solid phase material. The specifically bound protein of interest is then removed in active form from the immobilized ligand under appropriate conditions (e.g., low pH, high pH, ​​high salt, competing ligand, etc.) using an elution buffer and passed through the chromatography column without the contaminating proteins or protein impurities that were able to pass through the column earlier. Any component may be used as a ligand for purifying its respective specific binding protein, e.g., an antibody. However, in various methods according to the present disclosure, Protein A is used as a ligand for a target protein that includes an Fc region. Conditions for elution of a target protein (e.g., a protein that includes an Fc region) from a biospecific ligand (e.g., Protein A) can be readily determined by one of skill in the art. In some embodiments, Protein G or Protein L or functional variants thereof may be used as a biospecific ligand. In some embodiments, a biospecific ligand such as Protein A is used at a pH range of 5-9 for binding to a protein that includes an Fc region, with washing or re-equilibration of the biospecific ligand / target protein conjugate with a buffer having a pH of about 4 or less containing at least one salt, followed by elution.

[0042] The terms "purification," "separation," or "isolation," as used interchangeably herein, refer to increasing the purity of a protein of interest from a composition or sample that contains the protein of interest and one or more impurities. Typically, the degree of purity of the protein of interest 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 whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. Buffers resist changes in pH through the action of acid-base binding components. Buffers for use with biological reagents are generally capable of maintaining a constant concentration of hydrogen ions such that the pH of the solution is within the physiological range. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids and bases.

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

[0045] As used herein, the term "chromatography column" or "column" in reference to chromatography refers to a container, often in the form of a cylinder or hollow pillar, filled with a chromatography matrix or resin, which is a material that provides the physical and / or chemical properties used in purification.

[0046] The terms "ion exchange" and "ion exchange chromatography" refer to a chromatographic process in which an ionizable target solute (e.g., a target protein in a mixture) interacts (e.g., covalently) with oppositely charged ligands attached to a solid-phase ion exchange material under suitable pH and conductivity conditions, such that the target solute interacts nonspecifically with compounds that are more or less charged than the solute impurities or contaminants in the mixture. The contaminant solutes in the mixture may be washed off the column of ion exchange material or may bind to or be rejected from the resin faster or slower than the target solute. "Ion exchange chromatography" specifically includes cation exchange (CEX), anion exchange (AEX), and mixed-type chromatography.

[0047] "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 been passed over or through the solid phase. Any negatively charged ligand attached to a solid phase suitable for forming a cation exchange resin can be used, including, for example, 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 (GE Healthcare), TOYOPEARL® SP-650S and SP-650M (Tosoh), MACRO-PREP® High S (BioRad), Ceramic HyperD S, TRISACRYL® M and LS SP and Spherodex LS SP (Pall Technologies)); those with sulfoethyl-based groups (e.g., FRACTOGEL® SE (EMD), POROS® S-10 and S-20 (Applied Biosystems)); those with sulfopropyl-based groups (e.g., TSK Gel SP 5PW and SP-5PW-HR (Tosoh), POROS® HS-20, HS 50 and POROS® XS (Life Technologies); those with sulfoisobutyl-based groups (e.g., FRACTOGEL® EMD SO3 -(EMD); those having a sulfoxyethyl-based group (e.g., SE52, SE53, and Express-Ion S (Whatman)), those having a carboxymethyl-based group (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 (manufactured by JTBaker), SARTOBIND® S membrane (Sartorius), AMBERLITE® Strong Cation Exchangers, DOWEX® Strong Cation and DIAION® Strong Cation Exchanger (Sigma-Aldrich); or those with orthophosphate-based groups (e.g., P11 (Whatman)). Other cation exchange resins include carboxy-methyl-cellulose, BAKERBOND ABX™, Ceramic HyperD Z, Matrex Cellufine C500, Matrex Cellufine C200.

[0048] "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, including, for example, quaternary amino groups. Commercially available anion exchange resins include DEAE cellulose, POROS® PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 (from Applied Biosystems), SARTOBIND® Q (from Sartorius), MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance, QAE SEPHADEX® and FAST Q SEPHAROSE® (from GE Healthcare), WP PEI, WP DEAM, WP QUAT (from JT Baker), Hydrocell DEAE and Hydrocell QA (from Biochrom Labs Inc.), UNOsphere Q, MACRO-PREP® DEAE and MACRO-PREP® High Q (from Biorad), Ceramic HyperD Q, ceramic HyperD DEAE, TRISACRYL® M and LS DEAE, Spherodex LS DEAE, QMA SPHEROSIL® LS, QMA SPHEROSIL® M and MUSTANG® Q (Pall Technologies), DOWEX® Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX® MONOSPHER E 77, weak base anion (Dow Liquid Separations), INTERCEPT® Q Membrane, Matrex CELLUFINE® A200, A500, Q500 and Q800 (Millipore), FRACTOGEL® EMD TMAE, FRACTOGEL® EMDDEAE 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 (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] The term "flow-through" or "flow-through mode" as used herein refers to a general purification approach in which contaminants are removed from a mixture during chromatography because they are retained by the chromatographic process, usually by binding to a resin in a column. The protein of interest is purified because it does not bind (or binds less strongly than the contaminants) to the chromatographic medium (usually the resin in the column) and instead flows through and is collected. After elution of the protein of interest, the impurities bound to the column must be "stripped" or removed from the column so that the column can be regenerated for another chromatography run. This approach differs from "bind and elute" or "bind and elute mode" in which the target protein of interest is retained on the column and the impurities flow through the column. And this process involves the specific elution of the protein of interest using various column conditions that prevent the binding of the protein of interest to the chromatographic medium, usually the resin in the column.

[0050] The term "impurities" as used herein is used in its broadest sense to cover undesired components or compounds in a mixture. In cell culture, cell lysate 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 cell culture medium. Host cell impurity proteins include, but are not limited to, those naturally or recombinantly produced by host cells, as well as proteins (e.g., proteolytic fragments) related to or derived from the protein of interest and other process-related impurities. In certain embodiments, impurity precipitates are separated from cell culture using other means, such as centrifugation, sterile filtration, depth filtration and tangential flow filtration.

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

[0052] As used herein, "IgG antibodies", 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 be composed of two heavy chains (HC) and two light chains (LC), in which the two HCs and LCs are linked in the same number and positions of disulfide bridges that occur in naturally occurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody is mutated to change the disulfide bonds).

[0053] Immunoglobulins may be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. IgG isotypes are divided into subclasses in certain species, IgG1, IgG2, IgG3 and IgG4 in humans, and IgG1, IgG2a, IgG2b and IgG3 in mice. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by at most a few amino acids. "Antibody" includes, for example, both natural and non-natural antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies and completely synthetic antibodies.

[0054] The term "antigen-binding portion" of an antibody as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include: (i) a Fab fragment (fragment from papain cleavage) or similar monovalent fragments consisting of the VL, VH, LC and CH1 domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or similar bivalent fragments that contain two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single chain of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs that can be optionally linked by a synthetic linker. Furthermore, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but can be joined by a synthetic linker that can be engineered to produce a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (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-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced 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, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal of human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells, e.g., transfectomas, that have been transformed to express the antibody, (c) antibodies isolated from a recombinant combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by other means, including splicing human immunoglobulin gene sequences into other DNA sequences.

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

[0057] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0058] The term "polypeptide" as used herein 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 chain or chains of two or more amino acids and does not refer to a specific length of the product. The term "protein" as used herein is intended to encompass molecules composed of one or more polypeptides, which may optionally be linked by bonds other than amide bonds. On the other hand, a protein may also be one polypeptide chain. In this latter example, one polypeptide chain may optionally include two or more polypeptide subunits fused together to form a protein. The terms "polypeptide" and "protein" also refer to the product of post-expression modifications, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage or modification with non-naturally occurring amino acids. A polypeptide or protein may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be produced by any method, including chemical synthesis.

[0059] The term "polynucleotide" or "nucleotide" as used herein is intended to encompass single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), complementary DNA (cDNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide contains 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, e.g., DNA, cDNA, or RNA fragments. When applied to a nucleic acid or polynucleotide, the term "isolated" refers to a nucleic acid molecule, DNA, or RNA, that has been removed 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 that are maintained in heterologous host cells or that are purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. Polynucleotides or nucleic acids may also include regulatory elements, e.g., promoters, enhancers, ribosomal 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 over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where an identical nucleotide or amino acid is presented in both the target sequence and the reference sequence. 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 of the target sequence are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions where an identical amino acid residue or nucleic acid base is present 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. Comparison of sequences and determination of the percent sequence identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from a variety of sources for alignment of both protein and nucleotide sequences. 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 a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences and BLASTP is used to compare amino acid sequences.

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

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

[0064] The term "chase buffer" refers to a buffer used after the loading buffer to run a mixture or sample through a chromatographic 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 chromatography steps. In some embodiments, an AEX-CEX sequence is used and no pH and / or conductivity adjustment is made after the mixture leaves AEX and before it enters CEX. In other embodiments, a CEX-AEX sequence is used and no pH or conductivity adjustment is made after the mixture leaves CEX and before it enters AEX.

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

[0067] The term "LMW bodies" refers to any one or more unwanted proteins present in a mixture. Low molecular weight bodies 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 independent of the production of the intended protein of interest. Undesirable host cell proteins may be secreted into the upstream cell culture supernatant. Undesirable host cell proteins may 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 tightly to the Fc portion of immunoglobulins, and its use in purifying antibodies is well known in the art. Protein A has been used widely in the art for purification (Boyle et al., 1993; Hou et al. 1991). When applied to Protein A, the term "residual" or "rPA" refers to any remaining Protein A present in the mixture for use in further upstream purification of the protein of interest or antibody in the manufacturing process.

[0070] The term "buffer migration peak" refers to unwanted chromatographic elution due to change in conditions during chromatography. In some embodiments, the first buffer is a loading buffer used at the beginning of chromatography. In some embodiments, the second buffer is a chase buffer used to follow the mixture that is initially loaded through the chromatography process. Buffer migration peaks can appear during the transition from using a loading buffer to using a chase buffer. In other embodiments, buffer migration peaks can occur due to the difference between a first buffer, such as a loading buffer, and a second buffer, such as a chase buffer.

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

[0072] II. Purification method The present disclosure relates to a method for purifying a protein of interest 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 a flow-through mode under operating conditions where no changes to the operating conditions are made between AEX and CEX (or CEX and AEX). No changes to the operating conditions between AEX and CEX (or CEX and AEX) include, but are not limited to, automation, engineering control and / or in-line regulation. To avoid making changes between the processes of AEX and CEX (or CEX and AEX), in some embodiments, AEX and CEX (or CEX and AEX) can be contact-connected to each other as shown in FIG. 1. In other embodiments, AEX and CEX (or CEX and AEX) are not directly connected to each other, but the operating conditions (e.g., pH and / or conductivity) between AEX and CEX (or CEX and AEX) are not changed, i.e., are maintained in the same way. The operating conditions can be changed by the use of buffers during chromatography. In some embodiments, the chromatography comprises a loading buffer and / or a chase buffer.

[0073] In some embodiments, the combined AEX and CEX (or CEX and AEX) chromatography column is about 50 g / L resin, about 60 g / L resin, about 70 g / L resin, about 80 g / L resin, about 90 g / L resin, about 100 g / L resin, about 110 g / L resin, about 120 g / L resin, about 130 g / L resin, about 140 g / L resin, about 150 g / L resin, about 160 g / L resin, about 170 g / L resin, about 180 g / L resin, about 190 g / L resin, about 200 g / L resin, about 210 g / L resin, about 220 g / L resin, about 230 g / L resin, about 240 g / L resin, about 250 g / L resin, about 260 g / L resin, about 270 g / L resin, about 280 g / L 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. In some embodiments, the combined AEX and CEX (or CEX and AEX) chromatography column is loaded to about 300 g / L resin.

[0074] In some embodiments, when the pH of the buffer is different from the measured pI of the protein, the target protein in the present disclosure may carry a charge when placed in a buffer solution.When the target protein is found at a pH equivalent to its pI, it carries an overall neutral net charge.A target protein with a pI lower than the pH of the solution carries a net negative charge.Similarly, a target protein with a pI higher than the pH of the solution carries a net positive charge.

[0075] It is well known that the pH and ionic strength of the buffer are important for any form of ion exchange chromatography, so it is best to readjust the pH of the buffer after adjusting the salt concentration and ensure that the buffer counter ion is compatible. It is known that the buffer counter ion should have the same charge as the resin. In particular, when combining two opposite chromatographic steps together (e.g. AEX and CEX), it was expected that the pH and / or conductivity of AEX and CEX would be adjusted in between.

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

[0077] In some embodiments, the operating conditions of the 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, for example, a medium to high pI and a low pI. In some embodiments, the operating conditions are set for a target protein with a medium to high pI, for example, a pI≧6.5, and a target protein with a low pI, for example, a pI<6.5.

[0078] In some embodiments, the protein of interest has a medium to high pI, which is greater than about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0. In some embodiments, a protein of interest with a medium to high pI has a pI >= (greater than) 6.5 and a pI <= (less than) about 12.0, about 11.9, about 11.8, about 11.7, about 11.6, about 11.5, about 11.4, about 11.3, about 11.2, about 11.1, about 11.0, about 10.9, about 10.8, about 10.8, about 10.7, about 10.6, about 10.5, about 10.4, about 10.3, about 10.2, about 10.1 or about 10.0. In some embodiments, a protein of interest with a medium to high pI has a pI >= (greater than) 6.5 and a pI <= (less than) about 10.0, about 9.9, about 9.8, about 9.7, about 9.6, about 9.5, about 9.4, about 9.3, about 9.2, about 9.1, about 9.0, about 8.9, about 8.8, about 8.7, about 8.6, about 8.5, about 8.4, about 8.3, about 8.2, about 8.1 or about 8.0. In some embodiments, the target protein having a medium to high pI is about 7.0 to about 11.0, about 7.0 to about 10.9, about 7.1 to about 10.9, about 7.2 to about 10.9, about 7.2 to about 10.8, about 7.3 to about 10.8, about 7.3 to about 10.7, about 7.4 to about 10.7, about 7.4 to about 10.6, about 7.5 to about 10.6, about 7.5 to about 10.5, about 7.6 to about 10.5, about 7.6 to about 10.4, about 7.7 to about 10.4 , about 7.7 to about 10.3, about 7.8 to about 10.3, about 7.8 to about 10.2, about 7.9 to about 10.2, about 7.9 to about 10.1, about 8.0 to about 10.1, about 8.0 to about 10.0, about 8.1 to about 10.0, about 8.1 to about 9.9, about 8.2 to about 9.9, about 8.2 to about 9.8, about 8.3 to about 9.8, about 8.3 to about 9.7, about 8.4 to about 9.7, about 8.4 to about 9.6, about 8.5 to about 9.6, or about 8.5 to about 9.5. In other embodiments, the protein of interest 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, a protein of interest with a medium to high pI has a pI of about 7.0 to about 8.0, e.g., about 7.6. In other embodiments, a protein of interest with a medium to high pI has a pI of about 8.0 to about 9.0, e.g., about 8.4. In some embodiments, a protein of interest with a medium to high pI has a pI of 8.5 to 9.5, e.g., about 8.9. In other embodiments, a protein of interest with a medium to high pI has a pI of about 9.0 to about 10.0.

[0080] In some embodiments, the protein of interest has a low pI, which is less than 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, about 5.5, about 5.4, about 5.3, about 5.2, about 5.1 or about 5.0. In some embodiments, the protein of interest has a pI less than 6.5 and greater than about 1.0, about 2.0, about 3.0, about 4.0, about 5.0 or about 5.5. In some embodiments, a target protein having a low pI has a pI of about 1.0 to about 6.5, about 1.5 to about 6.5, about 2.0 to about 6.5, about 2.5 to about 6.5, about 2.5 to about 6.0, about 3.0 to about 6.5, about 3.0 to about 6.0, about 3.0 to about 5.5, about 3.5 to about 5.5, or about 3.5 to about 5.0.

[0081] In some embodiments, the protein of interest has an isoelectric point (pI) of about 4.8 to about 6.5, about 4.9 to about 6.5, about 4.9 to about 6.4, about 5.0 to about 6.4, about 5.0 to about 6.3, about 5.1 to about 6.3, about 5.1 to about 6.2, about 5.2 to about 6.2, about 5.2 to about 6.4, or about 5.3 to about 6.4. In other embodiments, the protein of interest has a pI of about 5.0 to about 6.0.

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

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

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

[0085] In some embodiments, the loading buffer of the combined chromatography 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.

[0086] In some embodiments, the protein of interest has a medium to high pI of about 7.0 to about 8.0 (eg, about 7.6) and the loading buffer for the protein has a pH of about 7.0 to about 8.0, eg, about 7.4.

[0087] In other embodiments, the protein of interest has a mid-high pI of about 8.0 to about 9.0 (eg, about 8.4) and the loading buffer for the protein has a pH of about 7.0 to about 8.0, eg, about 7.2.

[0088] In some embodiments, the protein of interest has a mid-high pI of 8.5 to 9.5 (eg, about 8.9) and the loading buffer for the protein has a pH of about 7.5 to about 8.5, eg, about 8.0.

[0089] In other embodiments, the protein of interest has a mid-high pI of about 9.0 to about 10.0 (eg, about 9.2) and the loading buffer for the protein has a pH of about 7.0 to about 8.0, eg, about 7.2.

[0090] In other embodiments, the protein of interest 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 the chase buffer for the combined chromatography have the same operating conditions. In certain embodiments, the 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) about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5 or about 10.0. In other embodiments, the chase buffer has a pH > (greater than) about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, about 10.2, about 10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about 10.9 or about 11.0.

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

[0094] In some embodiments, the chase buffer has a pH of about 6.0 to about 11.0, about 6.5 to about 11.0, about 7.0 to about 10.5, about 7.0 to about 10.0, about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, about 7.0 to about 8.0, about 7.0 to about 7.5, about 7.0 to 7.4, about 7.1 to about 7.4, or about 7.2 to about 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 protein of interest has a pI of about 7.0 to about 8.0, eg, 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, eg, about 7.6.

[0096] In other embodiments, the protein of interest 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 protein of interest has a pI of 8.5 to about 9.5, eg, about 8.9, and the loading buffer and chase buffer for the protein have the same pH of 8.5 to about 9.5, eg, about 8.9.

[0098] In other embodiments, the protein of interest has a pI of about 9.0 to about 10.0, such as 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, such as about 9.2.

[0099] In other embodiments, the protein of interest 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 the target protein with medium and high pI and the target protein with low pI can be different.In some embodiments, the conductivities for the target protein with medium and high pI and the target protein with low pI are different.In some embodiments, the conductivities for the protein with low pI are higher than the conductivities for the protein with medium to high pI.

[0101] In some embodiments, the conductivity for the loading buffer and / or chase buffer 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 the loading buffer and / or chase buffer 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 the loading buffer and / or chase buffer for a protein of interest having a medium to high pI is about 0.1 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 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 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm or about 9.0 mS / cm.

[0103] In some embodiments, the loading buffer and / or chase buffer for a protein of interest with 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, e.g., 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 a loading buffer and / or chase buffer for a protein of interest having a low pI is from about 6.0 mS / cm to about 15.0 mS / cm, from about 6.5 mS / cm to about 15 mS / cm, from about 7.0 mS / cm to about 14.0 mS / cm, from about 8.0 mS / cm to about 13.0 mS / cm, from about 8.0 mS / cm to about 12.0 mS / cm, or from about 9.0 mS / cm to about 12.0 mS / cm.

[0105] In some embodiments, the conductivity for a loading buffer and / or chase buffer for a protein of interest having a low pI is about 6.0 mS / cm, about 6.1 mS / cm, about 6.2 mS / cm, about 6.3 mS / cm, about 6.4 mS / cm, about 6.5 mS / cm, about 6.6 mS / cm, about 6.7 mS / cm, about 6.8 mS / cm, about 6.9 mS / cm, about 7.0 mS / cm, about 7.1 mS / cm, about 7.2 mS / cm, about 7.3 mS / cm, about 7.4 mS / cm, about 7.5 mS / cm, about 7.6 mS / cm, about 7.7 mS / cm, about 7.8 mS / cm, about 7.9 mS / cm, about 8 mS / cm, about 9 mS / cm, about 10 mS / cm, about 11 mS / cm, about 12 mS / cm, about 13 mS / cm, about 14 mS / cm, about 15 mS / cm, about 16 mS / cm, about 17 mS / cm, about 18 mS / cm, about 19 mS / cm, about 20 mS / cm, about 21 mS / cm, about 22 mS / cm, about 23 mS / cm, about 24 mS / cm, about 25 mS / cm, about 26 mS / cm, about 27 mS / cm, about 28 mS / cm, about 29 mS / cm, about 30 mS / cm, about 31 mS / cm, about 32 mS / cm, about 33 mS / cm, about 34 mS / cm, about 35 mS / cm, about 36 mS / cm, about 37 mS / cm, about 38 mS / cm, about 39 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, 14.8 mS / cm, about 14.9 mS / cm or about 15.0 mS / cm.

[0106] In some embodiments, the loading buffer and / or chase buffer for a protein of interest with 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 protein of interest has a pI of about 7.0 to about 8.0, e.g., 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, e.g., about 7.6, and a conductivity of 3.0 mS / cm to 6.0 mS / cm, e.g., 3.2 mS / cm and 5.5 mS / cm, respectively.

[0108] In other embodiments, the protein of interest has a pI of about 8.0 to about 9.0, e.g., 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, e.g., about 8.4, and a conductivity of 2.0 mS / cm to 4.0 mS / cm, e.g., 2.7 mS / cm and 3.0 mS / cm, respectively.

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

[0110] In other embodiments, the protein of interest has a pI of about 9.0 to about 10.0, e.g., 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, e.g., about 9.2, and a conductivity of 6.0 mS / cm to 7.0 mS / cm, e.g., 6.8 mS / cm and 6.8 mS / cm, respectively.

[0111] In other embodiments, the protein of interest has a pI of about 5.0 to about 6.0, e.g., 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, e.g., about 5.8, and a conductivity of 9.0 mS / cm to 12.0 mS / cm, e.g., 9.2 mS / cm and 12.0 mS / cm, respectively.

[0112] The methods (II and III) described herein can be used to purify proteins in a mixture containing contaminants including host cell proteins (HCPs), DNA, high molecular weight species (HMWs), low molecular weight species (LMWs), residual protein A (rPA), or any combination thereof. In some embodiments, the chromatography reduces (i) DNA to a level of about 20 pg / mL or less, (ii) LMWs to a level of about 1.0% or less, (iii) HMWs to a level of about 1.0% or less, (iv) HCPs 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, the chromatography reduces the DNA to levels of about 20 pg / mL or less, about 18 pg / mL or less, about 16 pg / mL or less, about 14 pg / mL or less, about 12 pg / mL or less, about 10 pg / mL or less, about 8 pg / mL or less, about 6 pg / mL or less, about 4 pg / mL or less, or about 2 pg / mL or less.

[0114] In some embodiments, the chromatography reduces low molecular weight species (LMW) to a level of 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.

[0115] In some embodiments, the chromatography reduces high molecular weight species (HMW) to a level of 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.

[0116] In some embodiments, the chromatography reduces host cell proteins (HCPs) to levels of 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.

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

[0118] In some embodiments, the method of the present disclosure is carried out after one or more downstream purifications, such as filtration, affinity chromatography, tangential flow chromatography, virus inactivation, or any combination thereof.In other embodiments, the method of the present disclosure is carried out without a post-harvest purification step.In other embodiments, the chromatography of the present disclosure is the last step before virus inactivation.In yet other embodiments, the method of the present disclosure simultaneously inactivates one or more viruses.In yet other embodiments, the method of the present disclosure further comprises virus inactivation before or after the combination of AEX and CEX or CEX and AEX.

[0119] III. Methods for removing buffer migration peaks The present disclosure also includes a method for reducing or eliminating buffer migration peaks between loading buffer and chase buffer.To reduce or eliminate buffer migration peaks in chromatography, such as AEX, CEX, a combination of AEX and CEX, or a combination of CEX and AEX, optionally in flow-through mode, the method includes adding a loading buffer and adding a chase buffer, where 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 chromatography.

[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, 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, at least 5. 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 other embodiments, the conductivity of the loading buffer is about 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 the conductivity 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 of reducing or removing the buffer migration peak (III) can be combined with the method of purifying a protein (II).Thus, in some embodiments, the present disclosure includes a method of purifying a protein of interest, comprising performing a combination of AEX and CEX or a combination of CEX and AEX chromatography without automation, engineering control and / or in-line adjustment between AEX and CEX, and under operating conditions (e.g., pH and / or conductivity) in which the chase buffer used in the chromatography has a higher conductivity than the loading buffer used in the chromatography.

[0123] Target Protein The method (II) and / or (III) of the present disclosure can be used to isolate or purify a protein of interest, such as a natural or non-natural protein, a recombinantly produced or plasma-purified protein, or any combination thereof. The protein of interest can be in a mixture with one or more contaminants as described elsewhere herein. In some embodiments, the protein of interest has already been subjected to downstream purification, such as filtration, affinity chromatography, tangential flow chromatography, viral inactivation, polishing, or any combination thereof. In other embodiments, the protein of interest is in a mixture with contaminants and has not been subjected to a post-harvest purification step.

[0124] In some embodiments, the mixture contains a protein of interest that is an antibody.

[0125] In some embodiments, the protein of interest is a fusion protein comprising the protein of interest 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, hesyl or sialylated protein.

[0126] In other embodiments, the protein of interest is produced in a host cell. In some embodiments, the protein of interest is produced in a culture comprising 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 fibroblast cells (COS-7), Madin-Darby bovine kidney cells (MDBK) or any combination thereof. In some embodiments, the starting mixture can be harvested cell culture fluid, 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 is passed through one or more downstream purification methods, such as diafiltration, ultrafiltration, affinity chromatography, tangential flow chromatography, virus inactivation, or any combination thereof.

[0128] The present disclosure is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0129] Example 1 Polishing Evaluation To evaluate the polishing step sequence and combination of polishing steps, as well as the effectiveness of reducing unwanted contaminants, various polishing schemes were examined, as shown in Table 1. The loading material for testing is the Protein A elution and neutralization pool (PAVIN) at pH 5.5 (without depth filtration). Before each run, the PAVIN pool is adjusted to pH 7.2 with 2M Tris. As shown in Table 1, CEX(F / T) can effectively remove HMW and rPA; AEX(F / T) can significantly reduce HCPs, but cannot reduce rPA and MW. With two-step polishing (CEX(F / T) followed by AEX(F / T)), HCPs, HMW, and rPA levels are all reduced to relatively low levels. Here, the combination of CEX-AEX F / T was also evaluated by connecting two columns and operating with CEX F / T conditions (load at pH 7.2 and 5 mS / cm, chase at pH 7.2 and 6 mS / cm). As shown in Figure 1, the outlet of the POROS XS column (10 cm bed height, 0.66 cm internal diameter) was directly connected to the inlet of a Capto Q column (10 cm bed height, 0.66 cm internal diameter) without any automation or engineering controls between the two columns.

[0130] To evaluate the effectiveness of the combined polishing step of CEX / AEX F / T without adjustment (AEX / CEX F / T), 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 different chromatographic approaches is detailed in Table 1. CEX alone, AEX alone, a combination of CEX-AEX with conductivity adjustment, and a combination of unadjusted CEX-AEX followed by AEX-CEX were tested under the operating conditions described above. All chromatography was operated in flow-through mode.

[0131] The combined CEX-AEX or AEX-CEXF / T polishing process without adjustments showed significant removal of HCPs, rPAs and HMWs, and reduced operation time / cost and skid footprint. Many advantages of combined CEX-AEX or AEX-CEXF / T polishing were shown; these include significant reduction in operation time, cost and skid footprint compared to a combined CEX(F / T) and AEX(F / T) polishing process with a single F / T polishing unit and conductivity adjustment between CEX and AEX. The combined CEX-AEX or AEX-CEX without pH and conductivity adjustments significantly increased downstream productivity compared to a single F / T polishing unit. Both CEX and AEX(F / T) operate at high loadings (300g / L or more). The combined CEX and AEX or AEX and CEX chromatography without pH and conductivity adjustments can utilize the same buffer system where CEX and AEX have the same charge, EQ, strip, purification and storage buffers. Modern chromatography can also be implemented by simply connecting the CEX outlet directly to the AEX inlet, as shown in Figure 1. [Table 1]

[0132] Example 2. Overview of molecules evaluated with CEX-AEX F / T polishing The purification effectiveness was measured for a number of antibodies with various isoelectric points (pI) and is detailed in Table 2. The first four antibodies were purified using a pH lower than the isoelectric point, indicating that the antibodies carry a slight positive net charge during chromatography. To prevent antibody binding to the column and facilitate run-through, salt was used to increase the conductivity of the buffer to a level where the antibody of interest was not strongly retained on the column, but unwanted contaminants such as HCP, rPA and HMW were retained. For the fifth antibody with pI 5.8, a higher salt concentration was used to prevent interaction of the negatively charged antibody with the anion exchange resin. [Table 2]

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

[0134] Example 3 Evaluation of Buffer Transition-Induced Post-Loading Peaks A protein mixture containing antibody-3 was passed through CEX(F / T) to evaluate its effectiveness in separating this antibody. CEX F / T was performed at pH 7.2 with 5 mS / cm loading buffer and 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 transition from loading buffer to chase buffer induced an unwanted post-buffer transition peak. When CEX F / T was performed again only by changing the conductivity of the chase buffer, i.e., 6 mS / cm, increased by 1 mS / cm, the chromatogram showed disappearance of the post-buffer transition peak, as shown in Figure 3B. Thus, by increasing the conductivity of the chase buffer, the peak due to the transition from loading buffer to chase buffer can be reduced or eliminated.

[0135] Throughout this application, various publications are referenced in parentheses by author name and date, or patent or patent publication number. The disclosures of these publications are incorporated herein by reference in their entirety in order to more fully describe the state of the art as known to those skilled in the art as of the date of disclosure described and claimed herein. The art of the art as known to those skilled in the art as of the date of disclosure described and claimed herein. However, the citation of a reference herein should not be construed as an admission that such reference is prior art to the present disclosure.

Claims

1. A method for purifying a protein from a mixture, 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, wherein no automation, engineering controls and / or in-line adjustments are used between AEX and CEX, and wherein pH and conductivity are not adjusted between AEX and CEX.

2. The method of claim 1 , wherein the AEX and the CEX are directly connected.

3. 3. The method of claim 1 or 2, wherein the protein has an isoelectric point (pI) greater than about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.

0.

4. 4. The method of claim 3, wherein the protein has a pI of less than about 12.0, about 11.9, about 11.8, about 11.7, about 11.6, about 11.5, about 11.4, about 11.3, about 11.2, about 11.1, about 11.0, about 10.9, about 10.8, about 10.7, about 10.6, about 10.5, about 10.4, about 10.3, about 10.2, about 10.1 or about 10.

0.

5. 5. The method of any one of claims 1 to 4, wherein 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.

6. 6. The method of any one of claims 1 to 5, wherein the protein has a pI of about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5 or about 11.

0.

7. 3. The method of claim 1 or 2, wherein the protein has a 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.

8. 8. The method of claim 7, wherein the protein has a pI greater than about 1.0, about 2.0, about 3.0, about 4.0 or about 5.

0.

9. 9. The method of claim 7 or 8, wherein 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.

10. 10. The method of claim 9, wherein 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.

11. The method according to any one of claims 1 to 6, wherein the combined chromatography comprises adding a loading buffer and adding a chasing buffer.

12. The method according to any one of claims 7 to 11, wherein the combined chromatography comprises adding a loading buffer and adding a chase buffer.

13. 13. The method of claim 11 or 12, wherein the loading buffer has a pH greater than about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5 or about 10.

0.

14. 14. The method of any one of claims 11 to 13, wherein the loading buffer has a pH of about 6.0 to about 11.0, about 6.5 to about 11.0, about 7.0 to about 10.5, about 7.0 to about 10.0, about 7.0 to about 9.5, about 7.0 to about 9.0, about 7.0 to about 8.5, about 7.0 to about 8.0 or about 7.0 to about 7.

5.

15. 14. The method of any one of claims 11 to 13, wherein the loading 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.

16. 16. The method of any one of claims 11 to 15, wherein the chase buffer has a pH greater than about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5 or about 9.

0.

17. 17. The method of any one of claims 11 to 16, wherein 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.

18. 17. The method of any one of claims 11 to 16, wherein the chase buffer has a pH of about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, 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 or about 9.

0.

19. The method of any one of claims 11 to 18, wherein the chase buffer has a pH that is the same or different from the pH of the loading buffer.

20. 20. The method of any one of claims 11 and 13-19, wherein the loading buffer has a conductivity of 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 to about 9.0 mS / cm, about 2.0 to about 8.0 mS / cm, about 2.0 to about 7.0 mS / cm, about 2.0 to about 8.5 mS / cm, about 2.0 to about 7.5 mS / cm or about 2.5 to about 7.0 mS / cm.

21. 21. The method of any one of claims 11 to 20, wherein the loading buffer has a conductivity of about 2.5 mS / cm to about 7.0 mS / cm.

22. 21. The method of any one of claims 11 to 20, wherein the loading buffer has a conductivity of about 0.1 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 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 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm or about 9.0 mS / cm.

23. 20. The method of any one of claims 12 to 19, wherein the loading buffer has a conductivity of about 6.0 mS / cm to about 15.0 mS / cm, about 6.5 mS / cm to about 15 mS / cm, about 7.0 mS / cm to about 14.0 mS / cm, about 8.0 mS / cm to about 13.0 mS / cm, or about 9.0 mS / cm to about 12.0 mS / cm.

24. 20. The method of any one of claims 12 to 19, wherein the loading buffer has a conductivity of about 9.0 to 9.5 mS / cm.

25. 20. The method of any one of claims 12 to 19, wherein the loading buffer has a conductivity of about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 10.5 mS / cm, about 11.0 mS / cm, about 11.5 mS / cm, about 12.0 mS / cm, about 12.5 mS / cm, about 13.0 mS / cm, about 13.5 mS / cm, about 14.0 mS / cm, about 14.5 mS / cm or about 15.0 mS / cm.

26. 23. The method of any one of claims 11 and 13-22, wherein the chase buffer has a conductivity of 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 to about 9.0 mS / cm, about 2.0 to about 8.0 mS / cm, about 2.0 to about 7.0 mS / cm, about 2.0 to about 8.5 mS / cm, about 2.0 to about 7.5 mS / cm or about 2.5 to about 7.0 mS / cm.

27. 23. The method of any one of claims 11 and 13-22, wherein the chase buffer has a conductivity of about 0.1 mS / cm, about 0.5 mS / cm, about 1.0 mS / cm, about 1.5 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 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 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm or about 9.0 mS / cm.

28. 26. The method of any one of claims 12-19 and 23-25, wherein the chase buffer has a conductivity of about 6.0 mS / cm to about 15.0 mS / cm, about 6.5 mS / cm to about 15.0 mS / cm, about 7.0 to about 15.0 mS / cm, about 8.0 to about 14.0 mS / cm, about 8.0 to about 13.0 mS / cm, about 8.0 to about 14.5 mS / cm, about 8.0 to about 13.5 mS / cm or about 8.5 to about 13.0 mS / cm.

29. 26. The method of any one of claims 12-19 and 23-25, wherein the chase buffer has a conductivity of about 6.0 mS / cm, about 6.5 mS / cm, about 7.0 mS / cm, about 7.5 mS / cm, about 8.0 mS / cm, about 8.5 mS / cm, about 9.0 mS / cm, about 9.5 mS / cm, about 10.0 mS / cm, about 10.5 mS / cm, about 11.0 mS / cm, about 11.5 mS / cm, about 12.0 mS / cm, about 12.5 mS / cm, about 13.0 mS / cm, about 13.5 mS / cm, about 14.0 mS / cm, about 14.5 mS / cm or about 15.0 mS / cm.

30. The method of any one of claims 11 to 29, wherein the conductivity of the chase buffer is higher, lower or the same as the conductivity of the loading buffer.

31. 31. The method of claim 30, 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.

32. The method of any one of claims 1 to 31, wherein the mixture comprises one or more contaminants.

33. 33. The method of claim 32, wherein the contaminants include host cell proteins (HCPs), DNA, high molecular weight matter (HMW), low molecular weight matter (LMW), residual protein A (rPA), or any combination thereof.

34. 34. The method of claim 33, wherein the 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.

35. 35. The method of claim 33 or 34, wherein the 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.

36. 36. The method of any one of claims 33 to 35, wherein the combined chromatography reduces the LMW 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.

37. 37. The method of any one of claims 33 to 36, wherein the combined chromatography reduces DNA levels to about 20 pg / mL or less, about 18 pg / mL or less, about 16 pg / mL or less, about 14 pg / mL or less, about 12 pg / mL or less, about 10 pg / mL or less, about 8 pg / mL or less, about 6 pg / mL or less, about 4 pg / mL or less, or about 2 pg / mL or less.

38. 38. The method of any one of claims 33 to 37, wherein the combined chromatography reduces the rPA level to about 6 ppm or less, about 5 ppm or less, about 4 ppm or less, about 3 ppm or less, about 2 ppm or less, about 1 ppm or less, about 0.8 ppm or less, about 0.6 ppm or less, about 0.5 ppm or less, about 0.4 ppm or less, about 0.3 ppm or less, about 0.2 ppm or less, or about 0.1 ppm or less.

39. 39. The method of any one of claims 11 to 38, 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 potassium, magnesium and calcium salts thereof, or any combination thereof.

40. 40. The method of claim 39, wherein the loading buffer comprises sodium acetate-Tris.

41. 41. The method of any one of claims 11 to 40, 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 potassium, magnesium and calcium salts thereof, or any combination thereof.

42. 42. The method of claim 41 , wherein the chase buffer comprises sodium acetate-Tris.

43. 43. The method of any one of claims 1 to 42, wherein the chromatography comprises a column loaded with up to 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.

44. Chromatography was performed using Poros HS, Poros XS, carboxy-methyl-cellulose, BAKERBOND ABX TM , sulfopropyl immobilized on agarose and sulfonyl immobilized on agarose, MonoS, MiniS, Source 15S, 30S, SP SEPHAROSE TM , C.M. 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 44. The method of any one of claims 1 to 43, comprising a CEX resin 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.

45. 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 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, 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 any combination thereof.

46. The method of any one of claims 1 to 45, wherein the protein is selected from an antibody or antibody fragment, a fusion protein, a natural protein, a chimeric protein, or any combination thereof.

47. 47. The method of claim 46, wherein the antibody is an isotype selected from IgM, IgA, IgE, IgD and IgG.

48. 48. The method of claim 47, wherein the IgG antibody is selected from IgG1, IgG2, IgG3 and IgG4.

49. 49. The method of any one of claims 1 to 48, wherein the chromatography inactivates viruses.

50. 50. The method of any one of claims 1 to 49, further comprising inactivating the virus.

51. 51. The method of claim 50, wherein the mixture is isolated by subjecting the starting mixture to affinity chromatography selected from Protein A affinity chromatography and Protein G affinity chromatography.

52. 52. The method of claim 51 , wherein the starting mixture is selected from a harvested cell culture fluid, a cell culture supernatant, a conditioned cell culture supernatant, a cell lysate, and a clarified bulk.

53. 53. The method of claim 51 or 52, wherein the starting mixture is derived from a mammalian cell culture.

54. 54. The method of claim 53, wherein the mammalian cell is a Chinese Hamster Ovary (CHO) cell.

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