Method for reducing endotoxin in protein sample

Abstract

The invention discloses a method for reducing endotoxin in a protein sample. The method comprises the following steps: carrying out affinity chromatography on a target protein-containing sample to be subjected to chromatography according to the following sequence: balancing, loading, post-balancing, flushing, leaching 1, leaching 2, eluting, collecting and post-treating to obtain a target protein crude product; and carrying out anion chromatography on the target protein crude product according to the following sequence: pre-balancing, balancing, loading a sample and collecting. The method is simple, efficient and stable, a sample with high endotoxin requirement can be prepared by using a conventional chromatographic filler in a laboratory, and extra expensive special filler expansion is not needed.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for reducing endotoxins in protein samples. Background Art

[0002] Proteins play a critical role in numerous fields, including biomedical research, drug development, and biotechnology. However, the presence of endotoxins in protein samples can seriously interfere with experimental results and even jeopardize the safety and efficacy of biological products. Endotoxins primarily originate from components of the cell wall of Gram-negative bacteria. When bacteria die, lyse, or are artificially disrupted, endotoxins are released into the environment, potentially contaminating protein samples. In studies of protein structure and function, endotoxins may interact with proteins, affecting their proper folding, aggregation, and activity assays. In the early stages of a project, the presence of endotoxins in biological products intended for human therapy, such as antibody drugs and recombinant proteins, can trigger serious adverse reactions and reduce drug safety and tolerability. Therefore, preparing protein samples low in endotoxins is a crucial task. During the production of biological products, endotoxins can be introduced through raw materials, production equipment, and the operating environment.

[0003] In addition to ensuring that all materials and reagents are endotoxin-free, endotoxin levels can also be reduced through affinity chromatography and anion chromatography. However, affinity ligands are expensive and may have nonspecific adsorption issues. In addition, affinity chromatography columns require regular maintenance and regeneration, and the operation is relatively complex. The effectiveness of anion chromatography in removing endotoxins may be affected by the properties of the protein and solution conditions, requiring optimization of experimental conditions. Moreover, for some proteins with similar charges to endotoxins, the separation effect may not be ideal.

[0004] Therefore, there is an urgent need in the art to develop new methods for reducing endotoxins in protein samples. Summary of the Invention

[0005] Based on this, it is necessary to provide at least a method for reducing endotoxins in protein samples.

[0006] In a first aspect of the present application, a method for reducing endotoxin in a protein sample is provided, the method comprising:

[0007] The sample containing the target protein to be chromatographed is subjected to affinity chromatography in the following order: equilibrium - sample loading - post-equilibrium - flushing - wash 1 - wash 2 - elution - collection, post-processing, to obtain the crude target protein;

[0008] The crude target protein was subjected to anion chromatography in the following order: pre-equilibration-equilibration-sample loading-collection.

[0009] In some embodiments, the affinity chromatography medium used in affinity chromatography is NMab Pro.

[0010] In some embodiments, the anion chromatography medium used in the anion chromatography is Diamond Q.

[0011] In some embodiments, the step of performing affinity chromatography comprises:

[0012] equilibrate at least the affinity chromatography column with affinity chromatography equilibration buffer;

[0013] Loading the sample to be chromatographed;

[0014] at least post-equilibrate the affinity chromatography column with affinity chromatography equilibration buffer;

[0015] At least flush the affinity chromatography column with affinity chromatography endotoxin removal buffer;

[0016] Elute the affinity chromatography column with affinity chromatography wash buffer 1 at least once;

[0017] at least eluting the affinity chromatography column with affinity chromatography wash buffer 2; and,

[0018] The affinity chromatography column is eluted with an affinity chromatography elution buffer, and samples with an affinity of 1000 mAu or more under UV280 are collected. The collected samples are then post-processed, including at least pH adjustment and filtration.

[0019] In some embodiments, prior to equilibration, line B is filled with affinity chromatography elution buffer.

[0020] In some embodiments, the step of performing affinity chromatography satisfies one or more of the following conditions 1) to 5):

[0021] 1) The affinity chromatography equilibration buffer comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 140 mM to 160 mM sodium chloride, and endotoxin-free water;

[0022] 2) the affinity chromatography elution buffer comprises 90 mM to 110 mM glycine and endotoxin-free water, with a pH of 3.40 to 3.60;

[0023] 3) Affinity chromatography endotoxin removal buffer contains: 10 mM–20 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.45 M–0.55 M arginine hydrochloride, and endotoxin-free water, pH 6.80–7.20;

[0024] 4) Affinity chromatography wash buffer 1 comprising 18 mM to 22 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.9 M to 1.1 M sodium chloride, and endotoxin-free water, pH 5.80 to 6.20; and,

[0025] 5) Affinity chromatography wash buffer 2 contains 18 mM–22 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, and endotoxin-free water, with a pH of 5.80–6.20.

[0026] In some embodiments, the step of performing anion chromatography comprises:

[0027] Pre-equilibrate at least the anion chromatography column with anion chromatography regeneration solution;

[0028] Equilibrate at least the anion chromatography column with anion chromatography equilibration solution;

[0029] loading the crude target protein; and,

[0030] Collect samples above 1000mAu under UV280.

[0031] In some embodiments, the loading flow rate is between retention time (RT) 5 min and RT 8 min.

[0032] In some embodiments, the A line and the anion chromatography column are pre-equilibrated with anion chromatography regeneration solution.

[0033] In some embodiments, the pre-equilibration is to a pH of 6.90 to 7.10.

[0034] In some embodiments, the A pipeline and the anion chromatography column are equilibrated with anion chromatography equilibration solution until the pH reaches 6.90-7.10 and the conductivity reaches 2.8 mS / cm-3.2 mS / cm.

[0035] In some embodiments, the anion chromatography equilibration solution comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and endotoxin-free water.

[0036] In some embodiments, the anion chromatography regeneration solution comprises 20 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 1 M to 1.1 M NaCl, and endotoxin-free water.

[0037] In some embodiments, prior to affinity chromatography, the chromatography system is fully disinfected with an alkaline solution and fully flushed with endotoxin-free water to remove the alkaline solution.

[0038] The method involved in this application is simple, efficient and stable, and can use conventional laboratory chromatography fillers to prepare samples with higher endotoxin requirements without the need for additional expensive special filler expansion; the preparation effect is good, and the anion can be reduced to below 1 Eu / mg after purification, which has good application value for the preparation of protein samples with low endotoxin requirements. DETAILED DESCRIPTION

[0039] To facilitate understanding of the present application, the present application will be described in more detail below. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0040] 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 application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0042] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0043] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0044] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0045] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0046] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0047] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0048] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0049] In this application, the term "first" in the "first aspect" is used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should it be understood as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first" serves only as a non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0050] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0051] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0052] Without wishing to be bound by any theory, it is believed that the competition between the charge interaction of arginine and endotoxin for binding sites and the negative charge exchange interaction of anion chromatography for the purpose of endotoxin removal can achieve better results.

[0053] In a first aspect of the present application, a method for reducing endotoxin in a protein sample is provided, the method comprising:

[0054] The sample containing the target protein to be chromatographed is subjected to affinity chromatography in the following order: equilibrium - sample loading - post-equilibrium - flushing - wash 1 - wash 2 - elution - collection, post-processing, to obtain the crude target protein;

[0055] The crude target protein was subjected to anion chromatography in the following order: pre-equilibration-equilibration-sample loading-collection.

[0056] Without wishing to be bound by any theory, it is believed that target proteins include biological products for human treatment, such as antibody drugs and recombinant proteins.

[0057] In some embodiments, the affinity chromatography medium used in affinity chromatography is, for example, NMab Pro, Protein A / G / L, etc.

[0058] In some embodiments, the anion chromatography medium used in the anion chromatography is Diamond Q.

[0059] In some embodiments, the step of performing affinity chromatography comprises:

[0060] S100: equilibrate at least the affinity chromatography column with an affinity chromatography equilibration buffer;

[0061] S200: loading the sample to be chromatographed;

[0062] S300: post-equilibrate the affinity chromatography column with an affinity chromatography equilibration buffer;

[0063] S400: flushing the affinity chromatography column with an affinity chromatography endotoxin removal buffer;

[0064] S500: eluting at least the affinity chromatography column with affinity chromatography elution buffer 1;

[0065] S600: eluting at least the affinity chromatography column with affinity chromatography elution buffer 2; and,

[0066] S700: Elute the affinity chromatography column with an affinity chromatography elution buffer, collect samples with an affinity of 1000 mAu or more under UV280, and perform post-processing on the collected samples including at least pH adjustment and filtration operations.

[0067] In some embodiments, in step S100, the A pipeline and the affinity chromatography column are equilibrated with an affinity chromatography equilibration buffer.

[0068] In some embodiments, in step S200, the sample to be chromatographed is loaded at a flow rate with a retention time of 5 min to a retention time of 8 min. For example, the sample to be chromatographed is loaded at a flow rate with a retention time of 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, 8 min, or a value or range between any two values.

[0069] In some embodiments, in step S300, the A pipeline and the affinity chromatography column are post-equilibrated with an affinity chromatography equilibration buffer.

[0070] In some embodiments, in step S400, line A and the affinity chromatography column are flushed with affinity chromatography endotoxin removal buffer. The flushing volume can be, for example, 5 column volumes or more; illustratively, the flushing volume is 6, 7, 8, 9, or 10 column volumes.

[0071] In some embodiments, in step S500, the A pipeline and the affinity chromatography column are eluted with affinity chromatography elution buffer 1.

[0072] In some embodiments, in step S600, the A pipeline and the affinity chromatography column are eluted with affinity chromatography elution buffer 2.

[0073] In some embodiments, in step S700, the affinity chromatography column is eluted with an affinity chromatography elution buffer, and a sample with an elution concentration of 1000 mAu or more under UV280 is collected. The collected sample is then post-processed, including at least pH adjustment and filtration, to adjust the pH to 6.90-7.10. Exemplarily, the pH is adjusted to 6.90, 6.95, 7.00, 7.05, 7.10, or any range or value between any two values.

[0074] In some embodiments, prior to equilibration, line B is filled with affinity chromatography elution buffer.

[0075] In some embodiments, the affinity chromatography equilibration buffer comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate (the sum of the concentrations of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate is 18 mM to 22 mM; unless otherwise specified, the total concentration of the two substances is referred to below), 140 mM to 160 mM sodium chloride, and endotoxin-free water.

[0076] Illustratively, the concentrations of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in the affinity chromatography equilibration buffer are 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, or a value or range between any two values.

[0077] Illustratively, the concentration of sodium chloride in the affinity chromatography equilibration buffer is 140 mM, 141 mM, 142 mM, 143 mM, 144 mM, 145 mM, 146 mM, 147 mM, 148 mM, 149 mM, 150 mM, 151 mM, 152 mM, 153 mM, 154 mM, 155 mM, 156 mM, 157 mM, 158 mM, 159 mM, 160 mM, or a value or range between any two values.

[0078] In some embodiments, the affinity chromatography elution buffer comprises 90 mM to 110 mM glycine and endotoxin-free water, with a pH of 3.40 to 3.60.

[0079] Illustratively, the concentration of glycine in the affinity chromatography elution buffer is 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, or a value or range between any two values.

[0080] Illustratively, the pH of the affinity chromatography elution buffer is 3.40, 3.45, 3.50, 3.55, 3.60, or a value or range between any two values.

[0081] In some embodiments, the affinity chromatography endotoxin removal buffer comprises: 10 mM to 20 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.45 M to 0.55 M arginine hydrochloride, and endotoxin-free water, with a pH of 6.80 to 7.20.

[0082] Illustratively, the concentration (total) of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in the affinity chromatography endotoxin removal buffer is 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, or a value or range between any two values.

[0083] Illustratively, the concentration of arginine hydrochloride in the affinity chromatography endotoxin removal buffer is 0.45 M, 0.46 M, 0.47 M, 0.48 M, 0.49 M, 0.50 M, 0.51 M, 0.52 M, 0.53 M, 0.54 M, 0.55 M, or a value or range between any two values.

[0084] Illustratively, the pH of the affinity chromatography endotoxin removal buffer is 6.80, 6.85, 6.90, 6.95, 7.00, 7.05, 7.10, 7.15, 7.20, or a value or range between any two values.

[0085] Illustratively, the affinity chromatography eluent 1 (also referred to as affinity chromatography eluent 1) comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, and endotoxin-free water.

[0086] In some embodiments, the affinity chromatography wash buffer 1 comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.9 M to 1.1 M sodium chloride, and endotoxin-free water, with a pH of 5.80 to 6.20.

[0087] In some embodiments, the concentration of sodium chloride in the affinity chromatography wash buffer 1 is 0.9 M, 1.0 M, 1.1 M, or a value or range between any two values.

[0088] In some embodiments, the pH of the affinity chromatography wash buffer 1 is 5.80, 5.90, 6.00, 6.10, 6.20, or a value or range between any two values.

[0089] In some embodiments, the concentration (total) of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in affinity chromatography wash buffer 1 is 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, or a value or range between any two values.

[0090] Illustratively, the affinity chromatography eluent 2 (also referred to as affinity chromatography eluent 2) comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, and endotoxin-free water.

[0091] In some embodiments, the affinity chromatography wash buffer 2 comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and endotoxin-free water, with a pH of 5.80 to 6.20.

[0092] In some embodiments, the concentration (total) of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in affinity chromatography wash buffer 2 is 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, or a value or range between any two values.

[0093] In some embodiments, the concentration of sodium chloride in the affinity chromatography wash buffer 2 is 0.9 M, 1.0 M, 1.1 M, or a value or range between any two values.

[0094] In some embodiments, the pH of the affinity chromatography wash buffer 2 is 5.80, 5.90, 6.00, 6.10, 6.20, or a value or range between any two values.

[0095] In some embodiments, the step of performing anion chromatography comprises:

[0096] Pre-equilibrate at least the anion chromatography column with anion chromatography regeneration solution;

[0097] Equilibrate at least the anion chromatography column with anion chromatography equilibration solution;

[0098] loading the crude target protein; and,

[0099] Collect samples above 1000mAu under UV280.

[0100] In some embodiments, the flow rate for loading the crude target protein is RT 5 min to RT 8 min. Exemplarily, the flow rate is RT 5 min, RT 5.5 min, RT 6 min, RT 6.5 min, RT 7 min, RT 7.5 min, RT 8 min, or a range or value between any two values.

[0101] In some embodiments, the A line and the anion chromatography column are pre-equilibrated with anion chromatography regeneration solution.

[0102] In some embodiments, the pH is pre-equilibrated to a pH of 6.90 to 7.10. Exemplarily, the pH is pre-equilibrated to a pH of 6.90, 6.95, 7.00, 7.05, 7.10, or a range or value between any two values.

[0103] In some embodiments, the A circuit and the anion chromatography column are equilibrated with anion chromatography equilibration solution to a pH of 6.90 to 7.10 and a conductivity of 2.8 mS / cm to 3.2 mS / cm. Exemplarily, the pH is 6.90, 6.95, 7.00, 7.05, 7.10, or a range or value between any two values. Exemplarily, the conductivity is 2.8 mS / cm, 2.9 mS / cm, 3.0 mS / cm, 3.1 mS / cm, 3.2 mS / cm, or a range or value between any two values.

[0104] In some embodiments, the anion chromatography equilibration fluid comprises 18 mM to 22 mM of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, and endotoxin-free water. Exemplarily, the total concentration of disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate in the anion chromatography equilibration fluid is 18 mM, 18.5 mM, 19 mM, 19.5 mM, 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, or a value or range between any two values.

[0105] In some embodiments, the anion chromatography regeneration fluid comprises 20 mM to 22 mM sodium phosphate dibasic dodecahydrate and sodium phosphate monobasic dihydrate, 1 M to 1.1 M NaCl, and endotoxin-free water. Exemplarily, the total concentration of sodium phosphate dibasic dodecahydrate and sodium phosphate monobasic dihydrate in the anion chromatography regeneration fluid is 20 mM, 20.5 mM, 21 mM, 21.5 mM, 22 mM, or any value or range between any two values.

[0106] In some embodiments, prior to affinity chromatography, the chromatography system is fully disinfected with an alkaline solution and fully flushed with endotoxin-free water to remove the alkaline solution.

[0107] Some examples are provided below.

[0108] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0109] Example 1

[0110] Before chromatography, thoroughly disinfect the chromatography system with 1M NaOH, ensuring that the system tubing and collection port are soaked in 1M NaOH for at least 1 hour. Then, thoroughly rinse the affinity chromatography medium NMab Pro with 0.2M NaOH for at least 30 minutes; and thoroughly rinse the Diamond Q anion chromatography medium with 1M NaOH for at least 30 minutes. Thoroughly rinse the system and chromatography column with endotoxin-free water to remove any residual alkaline.

[0111] Prepare six buffers: affinity chromatography equilibration buffer, affinity chromatography elution buffer, affinity chromatography endotoxin removal buffer, affinity chromatography eluent 1, affinity chromatography eluent 2, anion chromatography equilibration buffer, and anion chromatography regeneration buffer. Before use, disinfect the containers, stirring apparatus, pH and conductivity detection electrodes, and filter apparatus used in the preparation process by soaking in 1M NaOH and then thoroughly rinsing with endotoxin-free water.

[0112] 1. Buffer Preparation

[0113] Affinity chromatography equilibration buffer: 5.16 g disodium hydrogen phosphate dodecahydrate, 0.87 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride, and dilute to 1 L with endotoxin-free water.

[0114] Affinity chromatography elution buffer: glycine 7.51 g, dilute to 1 L with endotoxin-free water, and adjust the pH to 3.6 with 6 M HCl.

[0115] Affinity chromatography endotoxin removal buffer: 2.72 g disodium hydrogen phosphate dodecahydrate, 1.93 g sodium dihydrogen phosphate dihydrate, 105.33 g arginine hydrochloride, dilute to 1 L with endotoxin-free water, and adjust the pH to 7.0 with 4 M NaOH;

[0116] Affinity chromatography eluent 1: 0.43 g disodium hydrogen phosphate dodecahydrate, 2.93 g sodium dihydrogen phosphate dihydrate, 58.44 g sodium chloride, and dilute to 1 L with endotoxin-free water.

[0117] Affinity chromatography eluent 2: 0.88 g disodium hydrogen phosphate dodecahydrate, 2.74 g sodium dihydrogen phosphate dihydrate, and dilute to 1 L with endotoxin-free water.

[0118] Anion chromatography equilibrium solution: 2.40 g disodium hydrogen phosphate dodecahydrate, 0.52 g sodium dihydrogen phosphate dihydrate, and dilute to 1 L with endotoxin-free water.

[0119] Anion chromatography regeneration solution: 3.21 g disodium hydrogen phosphate dodecahydrate, 0.16 g sodium dihydrogen phosphate dihydrate, 58.44 g sodium chloride, and endotoxin-free water to 1 L.

[0120] 2. Chromatography

[0121] (1) Fill tubing B fully with affinity chromatography elution buffer until the pH is correct and the conductivity baseline is stable;

[0122] (2) Use affinity chromatography equilibration buffer to fully equilibrate line A and the affinity chromatography column to a pH value where the conductivity baseline is stable;

[0123] (3) Load the solution to be purified using line A at a flow rate of RT (Retention time): 5 min;

[0124] (4) Use affinity chromatography equilibration buffer to equilibrate line A and the affinity chromatography column to a pH value where the conductivity baseline is stable;

[0125] (5) Rinse line A and the affinity chromatography column with affinity chromatography endotoxin removal buffer for at least 5 Cv (column volume);

[0126] (6) Use affinity chromatography eluent 1 to flush pipe A and affinity chromatography column until the pH is adjusted and the conductivity baseline is stable;

[0127] (7) Use affinity chromatography eluent 2 to flush line A and the affinity chromatography column until the pH is adjusted and the conductivity baseline is stable;

[0128] (8) Use 100% affinity chromatography elution buffer to elute the target protein on the affinity chromatography column. The flow rate is controlled at RT: 5 min. Start collecting when the UV280 value reaches 1000 mAU and stop collecting when the UV280 value drops to 1000 mAU.

[0129] (9) Adjust the pH of the collected solution to 7.20 using a pH regulator (1 M Tris-base) and filter the collected solution using a disposable non-pyrogenic flat-top filter (0.22 μm);

[0130] (10) Use anion chromatography regeneration solution to fully balance the A pipeline and the anion chromatography column to pH 7.0, and the conductivity baseline is stable;

[0131] (11) Use anion chromatography equilibration solution to fully equilibrate pipeline A and anion chromatography column until the pH reaches 7.0 and the conductivity reaches 1.5 mS / cm;

[0132] (12) Use line A at a flow rate of RT: 5 min to load the sample to be collected by affinity chromatography;

[0133] (13) Start collecting when the UV280 value reaches 100 mAU. When the sample loading is completed, switch line A to the anion chromatography equilibrium solution. Stop collecting when the UV280 value drops to 1000 mAU.

[0134] The buffer solution used in the chromatography process should be sealed during use to avoid prolonged contact with air.

[0135] The results are shown in Tables 1 to 4. All samples used below are IgG1 monoclonal antibodies with an isoelectric point of 7.0-8.0.

[0136] Table 1

[0137]

[0138] Table 2

[0139]

[0140] Table 3

[0141]

[0142] Table 4

[0143]

[0144] Comparative Example

[0145] MabSelect PrismA was used as the affinity chromatography filler. Other conditions, such as the composition, dosage, and order of use of the affinity chromatography equilibration buffer, affinity chromatography elution buffer, and affinity chromatography wash buffer, were essentially the same as in Example 1. Affinity chromatography eluents 1 and 2 were also used for elution (step (6) in "2. Chromatography" was omitted). The collection strategy remained the same as in Example 1, and anion chromatography was not performed. The endotoxin test results of the samples harvested through this chromatography step are shown in Table 5 below (6.49 EU / mg).

[0146] Table 5

[0147]

[0148] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0149] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims, and the specification shall be used to interpret the content of the claims.

Claims

1. A method for reducing endotoxin in a protein sample, characterized in that: The method comprises: The sample containing the target protein to be chromatographed is subjected to affinity chromatography in the following order: equilibrium - sample loading - post-equilibrium - flushing - wash 1 - wash 2 - elution - collection, post-processing, to obtain the crude target protein; The crude target protein was subjected to anion chromatography in the following order: pre-equilibration-equilibration-sample loading-collection; The affinity chromatography medium used in affinity chromatography may optionally be NMab Pro; The anion chromatography medium used in anion chromatography may optionally be Diamond Q.

2. The method for reducing endotoxin in a protein sample according to claim 1, wherein The steps for affinity chromatography include: Equilibrating at least the affinity chromatography column with affinity chromatography equilibration buffer; optionally, equilibrating pipeline A and the affinity chromatography column; Loading the sample to be chromatographed; optionally, loading the sample at a flow rate with a retention time of 5 min to a retention time of 8 min; Post-equilibrate at least the affinity chromatography column with affinity chromatography equilibration buffer; optionally, post-equilibrate the A pipeline and the affinity chromatography column; flushing at least the affinity chromatography column with affinity chromatography endotoxin removal buffer; optionally, flushing the A line and the affinity chromatography column; the flushing volume is optionally at least 5 column volumes; Elute at least the affinity chromatography column with affinity chromatography wash buffer 1; optionally, elute pipeline A and the affinity chromatography column; Elute at least the affinity chromatography column with affinity chromatography wash buffer 2; optionally, elute pipeline A and the affinity chromatography column; and, The affinity chromatography column is eluted with affinity chromatography elution buffer, and samples with an affinity of 1000 mAu or more under UV280 are collected. The collected samples are post-processed by at least pH adjustment and filtration; optionally, the pH is adjusted to 6.90-7.

10.

3. The method for reducing endotoxin in a protein sample according to claim 2, wherein: Before equilibration, fill line B with affinity chromatography elution buffer.

4. The method for reducing endotoxin in a protein sample according to any one of claims 1 to 3, wherein: The affinity chromatography step satisfies one or more of the following conditions 1) to 5): 1) The affinity chromatography equilibration buffer comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 140 mM to 160 mM sodium chloride, and endotoxin-free water; 2) the affinity chromatography elution buffer comprises 90 mM to 110 mM glycine and endotoxin-free water, with a pH of 3.40 to 3.60; 3) Affinity chromatography endotoxin removal buffer contains: 10 mM–20 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.45 M–0.55 M arginine hydrochloride, and endotoxin-free water, pH 6.80–7.20; 4) Affinity chromatography wash buffer 1 comprising 18 mM to 22 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, 0.9 M to 1.1 M sodium chloride, and endotoxin-free water, pH 5.80 to 6.20; and, 5) Affinity chromatography wash buffer 2 contains 18 mM–22 mM sodium phosphate dibasic dodecahydrate and sodium dihydrogen phosphate dihydrate, and endotoxin-free water, with a pH of 5.80–6.

20.

5. The method for reducing endotoxin in a protein sample according to any one of claims 1 to 3, wherein: The steps for performing anion chromatography include: Pre-equilibrate at least the anion chromatography column with anion chromatography regeneration solution; Equilibrate at least the anion chromatography column with anion chromatography equilibration solution; The crude target protein is loaded, and the loading flow rate can be optionally from retention time 5 min to retention time 8 min; and, Collect samples above 1000mAu under UV280.

6. The method for reducing endotoxin in a protein sample according to claim 5, wherein: Use anion chromatography regeneration solution to pre-equilibrate pipeline A and anion chromatography column; Optionally, pre-equilibrate to a pH of 6.90-7.

10.

7. The method for reducing endotoxin in a protein sample according to claim 5, wherein: Use anion chromatography equilibration solution to equilibrate pipeline A and anion chromatography column until the pH reaches 6.90~7.10 and the conductivity reaches 2.8mS / cm~3.2mS / cm.

8. The method for reducing endotoxin in a protein sample according to claim 5, characterized in that: The anion chromatography equilibrium solution comprises 18 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, as well as endotoxin-free water.

9. The method for reducing endotoxin in a protein sample according to claim 5, characterized in that: The anion chromatography regeneration solution comprises 20 mM to 22 mM disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate, 1 M to 1.1 M NaCl, and endotoxin-free water.

10. The method for reducing endotoxin in a protein sample according to any one of claims 1 to 9, wherein: Before affinity chromatography, the chromatography system should be fully disinfected with alkaline solution and flushed with endotoxin-free water to remove the alkaline solution; Optionally, soak the tubing and collection port of the chromatography system with a NaOH solution having a concentration of at least 1 M; Optionally, wash the affinity chromatography medium with a NaOH solution having a concentration of at least 0.2 M for at least 30 min; Optionally, wash the anion chromatography medium with a NaOH solution of at least 1 M concentration for at least 30 min.

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