CN113155823A

CN113155823A - Method for characterizing degradation activity of esterase in host cell protein on polysorbate

Info

Publication number: CN113155823A
Application number: CN202110556872.6A
Authority: CN
Inventors: 王伟文; 袁梵雨; 陈全民; 郭哲明
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd
Priority date: 2021-05-21
Filing date: 2021-05-21
Publication date: 2021-07-23

Abstract

Disclosed herein is a method of characterizing the polysorbate degrading activity of an esterase on a host cell protein, the method comprising: a) reacting a sample comprising host cell proteins with naphthyl acetate; b) carrying out color development reaction on a reaction product of the sample and naphthyl acetate and a fast blue B salt; and c) detecting the absorbance of the chromogenic reaction product at 550nm to 600 nm; the increase in absorbance caused by the reaction of the sample with naphthyl acetate indicates the presence of degradation activity of the polysorbate by the esterase in the host cell protein in the sample. The method is convenient, rapid, low in cost and high in sensitivity and is used for representing the degradation activity of the esterase in the host cell protein on the polysorbate.

Description

Method for characterizing degradation activity of esterase in host cell protein on polysorbate

Technical Field

The application belongs to the technical field of biological medicines, and particularly relates to a quantifiable method for representing degradation activity of esterase in host cell protein on polysorbate.

Background

The biological medicine technology is the core strength of the development of the current medicine technology. Biological products typified by monoclonal antibody drugs are important drug classes for treating diseases such as malignant tumors, autoimmune diseases, and alzheimer's disease.

Polysorbates such as polysorbate 20 and 80 are the most commonly used surfactants in biopharmaceuticals, and they can improve the stability of protein formulation drugs and prevent protein aggregation and denaturation during long-term storage and transportation of protein formulation drugs, thereby preventing degradation of the quality of protein formulation drugs. However, polysorbate is easily hydrolyzed under certain conditions, which causes protein aggregation and causes the quality of the medicine to be reduced. The hydrolysis mechanism of polysorbate has been extensively studied in industry, and in addition to the induced hydrolysis of acids and bases, Host Cell Protein (HCP) is co-purified with target protein and finally remains in protein preparations, which is the most important cause of polysorbate hydrolysis. Because the means for detecting HCP is deficient in the early development process of protein preparation medicines, the research on the influence of HCP on the degradation condition of polysorbate has to be omitted in the development process of protein preparation medicines, and as a result, the aggregation and denaturation of protein are easily caused in the long-term storage and transportation process of protein preparation medicines, and more resources are consumed in the later investigation and research for solving the problem of medicine quality degradation. For the above reasons, there is a need for a method for detecting the degradation activity of an esterase in HCP on polysorbate in an early stage of protein preparation drug development, conveniently, rapidly and with high sensitivity.

Disclosure of Invention

To address the above-described problems in the prior art and to meet the needs in the art, the present invention provides a method for characterizing the polysorbate-degrading activity of an esterase in a host cell protein, which is quantifiable. Specifically, the invention provides a method for characterizing the polysorbate degrading activity of esterase in host cell proteins, which is characterized by comprising the following steps:

a) reacting a sample comprising host cell proteins with naphthyl acetate;

b) carrying out color development reaction on a reaction product of the sample and naphthyl acetate and a fast blue B salt; and

c) detecting the absorbance of the chromogenic reaction product at a wavelength of 550nm to 600 nm;

the increase in absorbance caused by the reaction of the sample with naphthyl acetate indicates the presence of degradation activity of the polysorbate by an esterase in the host cell protein in the sample.

Wherein, the sample can be intermediate products collected from each section and each node after the culture and harvest of the host cells, and can also be final products after final purification. The method can also determine the enzyme activity, specific activity and/or unit volume activity of the esterase in the sample so as to quantitatively characterize the degradation activity of the esterase in the host cell protein on the polysorbate.

The method can quantitatively analyze whether the intermediate product has esterase activity capable of degrading polysorbate. The method of the invention can be used in the development of downstream purification processes, and guides the development of purification processes, in particular processes for removing esterase from host cell proteins, by quantifying the decrease in esterase activity with the optimization of the downstream processes. The method of the invention can be used to quantify the activity of residual esterase in the final product, e.g. stock or finished product, guiding the assessment of the risk of degradation of polysorbate by HCP.

Drawings

FIG. 1: in one example, a standard curve of naphthol content versus absorbance. Wherein the abscissa is the measured absorbance and the ordinate is the naphthol content.

FIG. 2: in one example, polysorbate 80 competitively inhibits the mie equation double reciprocal plot of esterase activity for naphthyl acetate degradation. Wherein the abscissa is 1/[ S ] and the ordinate is 1/v.

Detailed Description

As used herein, unless otherwise specified, the singular forms "a", "an", and "the" preceding singular forms "a", "an", and "the" include plural referents.

Herein, unless otherwise specified, the technical features mentioned herein, such as components, contents, steps, condition parameters, parameter values, components, connections, working relationships, and the like, are not limited to the embodiments and examples specifically described herein, and any other combinations are also within the scope of the present invention.

Herein, unless otherwise specified, numerical ranges expressed as two end values are to be considered as specifically disclosing both end values and all real numbers between the end values and their combined numerical ranges. Furthermore, unless specifically stated otherwise, when a plurality of alternative ranges or values are specified for the same feature or the same parameter, these values and limits may be arbitrarily combined, and the ranges derived therefrom are intended to fall within the scope of the present invention as long as they are contained within the maximum continuum specified.

Herein, unless otherwise specified, the numerical values, whether with the antecedent "about", encompass the approximate range of equivalence to the numerical value that can be understood by those skilled in the art, such as the range of the numerical value ± 10%, 5%, 3%, 2%, 1%, or ± 0.5%.

Herein, unless otherwise specified, all scientific and technical terms have the meaning according to what is known or known in the art, in particular in the field of recombinant protein production and development, for example as described in textbooks, laboratory manuals or prior art documents.

In this context, unless otherwise specified, percentages (%) with respect to the solution system are mass to volume ratios (w/v) and the ratio between solution and solution is volume ratio. "solution" refers broadly to a multi-component liquid system unless otherwise specified.

The inventor finds that esterase capable of degrading naphthyl acetate and polysorbate in HCP belongs to the same enzyme system, so naphthol is generated by utilizing the degradation reaction of the esterase in HCP on naphthyl acetate, the absorbance of a chromogenic reaction product is detected by utilizing the chromogenic reaction of fast blue B salt and naphthol, and the absorbance is increased after a period of reaction time, so that the degradation activity of HCP on polysorbate is represented. The method can be used for detecting the esterase activity in the HCP in the early stage of protein preparation drug research, so that the downstream purification process is fully optimized to remove the residual esterase in the product in the early stage of research and development of the drug, the problem of quality reduction generated in the process of transportation or long-term storage after the production of the protein preparation drug is avoided as much as possible, and the method can be used for quantitatively detecting the esterase activity in the final product to evaluate the risk of polysorbate reduction caused by the esterase.

Specifically, the present invention provides a method for characterizing the polysorbate degrading activity of an esterase in a Host Cell Protein (HCP), said method comprising:

a) reacting a sample comprising host cell proteins with naphthyl acetate;

In the method, the sample can be a sample collected from each section and each node in the process flow of producing the protein product by the recombinant method, and comprises a sample of an intermediate product and a sample of a final product. In some embodiments, the methods of the invention detect the polysorbate degrading activity of an esterase in a host cell protein in the intermediate product. Herein, "intermediate" includes products obtained by processes of producing protein-based products by recombinant methods starting from starting materials through various processes such as fermentation, culture, isolation and purification, addition of necessary stabilizers, and the like (Chinese pharmacopoeia 2015 edition). For example, intermediates include, but are not limited to, harvested supernatants after fermentation and products obtained from downstream processing such as separation, purification, concentration, formulation, etc. Examples of separation and purification include, but are not limited to, clarification (e.g., centrifugation, filtration, etc.) and chromatography (e.g., affinity, ion exchange, hydrophobic, multimodal, etc.).

In other embodiments, the methods of the invention detect the polysorbate degrading activity of an esterase in a host cell protein in the final product. Herein, "end product" includes a stock or finished product, such as a stock or finished product of a recombinant protein (e.g., a monoclonal antibody). Wherein, the 'stock solution' refers to the harvest solution which is extracted, purified and subpackaged in an intermediate storage container to obtain the stock solution; the finished product refers to the finished product obtained by sterilizing and filtering the raw liquid or semi-finished product, subpackaging in a sterile final container and packaging (2015 edition of Chinese pharmacopoeia).

In some embodiments, step a) the naphthyl acetate is contained in a substrate solution and reacted with the sample. The substrate solution may comprise: about 0.01 to 0.2mol/L, about 0.04 to 0.1mol/L, or about 0.04mol/L of a disodium hydrogen phosphate/sodium dihydrogen phosphate buffer at a pH of about 6.0 to 8.0, preferably at a pH of about 7.0; about 0.5-3% (v/v), about 1-2% (v/v), or about 1% (v/v) acetone; and about 0.00001 to 0.006mol/L, preferably about 0.0001 to 0.003mol/L, such as about 0.0003mol/L, about 0.001mol/L of naphthyl acetate.

In some embodiments, the temperature for reacting the sample with the naphthyl acetate substrate in step a) may be about 25 to 30 ℃ and the incubation time may be about 0.5 to 120 hours. One skilled in the art will appreciate that the relative magnitude of the enzyme activity can be estimated based on the source of the sample, and that the reaction temperature and incubation time can be optimized based on the relative estimated enzyme activity. For example, in some embodiments, the sample being assayed is an intermediate product, such as a harvest supernatant or a chromatographically purified product, and the incubation can be for about 0.5 to 6 hours, such as about 1, about 2, about 3, about 4, about 5 hours; in other embodiments, the sample to be tested is a final product, such as a recombinant protein (e.g., monoclonal antibody) stock solution and a finished product, and the incubation time can be about 6 to 120 hours, such as about 6 to 72 hours, such as about 6, 24, 72 hours.

In some embodiments, in step B), the fast blue B salt as developer is contained in a solution, which is referred to herein as "color former". The color developing solution may further contain sodium lauryl sulfate. For example, the color developing solution may be a mixture of a 5% (w/v) sodium lauryl sulfate solution and a 1% (w/v) fast blue B salt solution in a volume ratio of about 10:1 to 1:1, for example, about 5: 2. The solvent of the above-mentioned solution may be water, such as ultrapure water.

Herein, "color reaction product" refers to a color reaction system after undergoing a color reaction. Herein, the "color development reaction system" refers to a mixed system of a sample (e.g., naphthol or naphthol solution in the case of preparing a standard curve) in which naphthol is a color development substrate or a sample to be tested for the presence of naphthol (e.g., a reaction product of a sample containing HCP and naphthyl acetate), and a color developer or a color developing solution. Herein, the "absorbance of the color-developing reaction product" is simply referred to as "absorbance" in the context.

In some embodiments, the color reaction in step b) comprises centrifuging the color reaction system at about 4000 to 10000rpm, such as about 8000rpm, for about 5 to 10 minutes. Then, the supernatant obtained by centrifugation was taken to measure absorbance. In some embodiments, the sample is the final product and the chromogenic reaction comprises centrifugation at about 8000 to 10000rpm, for example, about 8000rpm, for about 5 to 10 minutes. In some embodiments, the reaction product of the HCP-containing sample and naphthyl acetate is centrifuged immediately after mixing with the developer or color former, and the supernatant is taken immediately after centrifugation to detect absorbance.

In some embodiments, the volume ratio of sample to substrate solution is about 1:1 to about 1:10, e.g., about 1: 5. In some embodiments, the ratio of the color developing solution to the sample is about 1:1 by volume. In some embodiments, the volume ratio of substrate solution to sample to developing solution is about 5:1: 1.

In the method of the present invention, the increase in absorbance caused by the reaction of the sample with naphthyl acetate is understood to mean that the increase in absorbance as the reaction proceeds is observed when the absorbance at the rear end of the reaction is compared with the absorbance at the front end of the reaction for any length of time during the reaction. The time point of absorbance detection is not limited. For example, in some embodiments, the absorbance may be measured as the initial absorbance immediately after the sample is mixed with the naphthyl acetate substrate (also referred to as "at 0"), or may be measured at a later time; the absorbance at the latter time or "absorbance after reaction" may be the absorbance measured at any time (e.g., the end of the reaction) after the initial absorbance measurement; an increase in absorbance after the reaction compared to the initial absorbance indicates the presence of degradation activity of the polysorbate by esterase in the host cell protein in the sample. The determination of these points in time is freely determinable by the skilled person.

In some embodiments, the method further comprises determining the enzymatic activity, specific activity, and/or unit volume activity of the esterase in the sample from the measured absorbance to quantitatively characterize the degradation activity of the esterase on polysorbates in the host cell protein. For example, the yield of naphthol, which is an enzymatic hydrolysis product, can be determined by making and using a standard curve of absorbance and naphthol concentration, and quantitative data such as enzyme activity, specific activity and unit volume activity can be determined by combining sample volume, protein content and the like.

The method of the present invention is suitable for the recombinant production process of various protein biological medicines by using various host cells. For example, the host cell may be a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell. For example, the recombinant protein may be a monoclonal antibody and a functional fragment thereof, a genetically engineered antibody and a functional fragment thereof, a non-antibody immunoglobulin, an Fc fusion protein, and any recombinant protein without a fusion tag, such as a recombinase protein.

The method of the invention is suitable for characterizing the degradation activity of esterases in HCPs on various polysorbates, such as polysorbate 20(PS20) and polysorbate 80(PS80), which are most commonly used in biopharmaceutical formulations.

Examples

The technical solution of the present invention will be described in more detail with reference to specific examples. The following examples are merely illustrative and do not limit or restrict the technical solutions of the present invention. The following examples are illustrative, but not exhaustive, of specific materials, steps, conditions, values, or ranges of values and other technical parameters.

In the following examples, all solvents used for preparation were ultrapure water unless otherwise specified. In the following examples, "naphthyl acetate" refers to alpha-naphthyl acetate. In the following examples, the HCP concentration was measured using the Cygnus F550 CHO HCP ELISA kit (Cygnus Co.). In the following examples, "ppm" means "mg/L" unless otherwise specified.

Example 1: detection of esterase specific activity in intermediate product

Standard curve

A standard curve was made as follows:

preparing a solution diluent comprising 0.04mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate buffer, pH 7.0, 1% acetone;

preparing a naphthol standard solution, wherein the naphthol standard solution contains 0.04mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate buffer solution, pH 7.0, 1% acetone and 0.006mol/L naphthol;

preparing a color developing solution: mixing 5% sodium dodecyl sulfate solution and 1% fast blue B salt solution according to the ratio of 5:2 volume ratio.

Using dilute solutionDiluting the naphthol standard solution by the release agent in proportion, and mixing the diluted naphthol standard solution with the developing solution according to the ratio of 6: 1, detecting the absorbance of the chromogenic reaction product, and drawing a standard curve by using the measured data with the absorbance as an abscissa and the naphthol content as an ordinate, as shown in table 1 and fig. 1. Standard curve equation: y 1.3687x +0.176, R²＝0.9986。

Table 1: standard curve data

Naphthol content (. times.10)^-3μmol)	1.5	3.0	3.75	5.0	7.5	15
							Absorbance at 600nm	0.79	2.04	2.50	3.74	5.35	10.80

Activity detection

In this example, the intermediate productSamples were taken from the intermediate solutions obtained after the supernatant harvested after cell culture of CHO cells was subjected to capto blue (high sub) affinity chromatography. HCP concentration in the sample was 1.0X 10³ppm。

The substrate solution was prepared as 0.04mol/L disodium hydrogenphosphate/sodium dihydrogenphosphate buffer pH 7.0, 1% acetone, 0.0003mol/L alpha-naphthyl acetate. A reaction mixture (600. mu.L) was prepared in duplicate by mixing 100. mu.L of the sample with 500. mu.L of the prepared substrate solution. A portion of the reaction mixture was taken as a substrate for color development reaction and immediately subjected to color development reaction and absorbance was measured and recorded as "at 0" value. The other reaction mixture was incubated at 25 ℃ for 0.5 hour to give a reaction product solution, and then a color development reaction was carried out using the reaction product solution obtained by the incubation as a color development reaction substrate and absorbance was measured.

Preparing a fresh color developing solution: 500. mu.L of 5% sodium lauryl sulfate solution and 200. mu.L of 1% fast blue B salt solution were mixed well. The color developing solution is prepared and used immediately before use.

And (3) color development reaction: 100. mu.L of the color-developing solution was mixed with the color-developing reaction substrate, and centrifuged at 8000rpm for 10 minutes. The supernatant from the centrifugation was then taken and the absorbance at 600nm was measured, the results are shown in the following table:

table 1: the result of the detection

	At 0 time	0.5 hour
			Absorbance at 600nm	2.35	5.28

And (3) obtaining the naphthol content serving as an enzymolysis product in the reaction product solution according to the measured absorbance by using a standard curve, and calculating the specific activity and unit volume activity of the esterase in the HCP according to the naphthol yield and the sample volume and HCP concentration. Wherein the content of the first and second substances,

the unit of enzyme activity is defined as: the amount of enzyme that catalyzes the conversion of 1 μmol of substrate in 1 minute under standard reaction conditions is defined as one enzyme activity unit (U), i.e. 1U ═ 1 μmol/min;

specific Activity (SA): the number of units of enzyme activity per mg of esterase protein;

activity per unit volume: the enzyme activity per ml of the solution is the unit number.

In this example, the volume activity of esterase in HCP in the intermediate product obtained by affinity purification was calculated to be 0.67U/mL of the product solution, and the specific activity was calculated to be 0.671U/mg of HCP esterase protein.

Therefore, the method can quantitatively describe the specific activity of the esterase, and the esterase is a key enzyme for degrading the polysorbate in the HCP, so the specific activity can be used for quantitatively evaluating the degrading activity of the esterase on the polysorbate in the HCP. During process development, the status of esterase clearance in HCP can be assessed by specific activity and the risk of degradation of polysorbate by product solution by unit volume activity.

Example 2: detection of esterase specific activity in end product

Standard curve

A standard curve was made as described in example 1.

Activity detection

In this example, the final product sample was obtained from the CHO-produced monoclonal antibody, wherein the HCP concentration was 10.4 ppm.

The substrate solution was prepared as 0.04mol/L disodium hydrogenphosphate/sodium dihydrogenphosphate buffer pH 7.0, 1% acetone, 0.0003mol/L alpha-naphthyl acetate. Duplicate reaction mixtures (600. mu.L) were prepared by mixing 100. mu.L of the final solution with 500. mu.L of the prepared substrate solution. A portion of the reaction mixture was taken as a substrate for color development reaction and immediately subjected to color development reaction and absorbance was measured and recorded as "at 0" value. The other part of the reaction mixture was incubated at 30 ℃ for 120 hours to obtain a reaction product solution, and then a color development reaction was carried out using the reaction product solution obtained by incubation as a color development reaction substrate and absorbance was measured.

And (3) color development reaction: 100. mu.L of the color-developing solution was mixed with the color-developing reaction substrate, centrifuged at 8000rpm for 10 minutes, and the supernatant obtained by the centrifugation was measured for absorbance at 600nm, with the results shown in the following Table:

table 2: the result of the detection

	At 0 time	120 hours
			Absorbance at 600nm	0.34	5.97

The volume activity of the esterase in the finished product in this example was calculated to be 0.01U/mL of product solution and the specific activity was calculated to be 0.532U/mg HCP esterase protein. As can be seen from comparison with the results of example 1, the finished solution is far less at risk of degradation of PS80 than the intermediate solution.

Example 3: characterization of degradation Activity of naphthyl acetate on polysorbate degradation Activity

Experiment I polysorbate 80 competitively inhibits the degradation activity of esterase to naphthyl acetate

Competitive experimental sample preparation:

preparing a solution diluent: 0.04mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate buffer, pH 7.0, 1% (v/v) acetone;

preparing a reaction substrate solution, wherein the reaction substrate solution comprises 0.04mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate buffer solution, pH 7.0, 1% (v/v) acetone and 0.003mol/L alpha-naphthyl acetate;

preparing a 10% polysorbate 80 mother liquor, wherein the polysorbate 80 mother liquor comprises 10%;

preparing a color developing solution: the 5% sodium lauryl sulfate solution was mixed with 1% fast blue B salt solution at a volume ratio of 5: 2.

Polysorbate 80 and control groups were set up separately and the substrate solutions were diluted in a gradient as shown in table 4. The activity was measured as follows:

control group: mu.L of the intermediate sample described in example 1 was mixed with 500. mu.L of each dilution of the substrate solution, and incubated at 25 ℃ for 0.5 hour to obtain a reaction product solution. 100. mu.L of the color developing solution was mixed with the reaction product solution (600. mu.L), and centrifuged at 8000rpm for 10 minutes. The supernatant from the centrifugation was then taken and the absorbance at 600nm was measured.

The polysorbate 80 group differed from the control group only in that: samples of the intermediate described in example 1 were taken to formulate 100 μ L of a test sample containing another 0.04% polysorbate 80 solution with 10% polysorbate 80 stock solution and reacted in a mixture of 500 μ L of each dilution of substrate solution.

The results are shown in the following table:

table 4: competitive inhibition experiment detection result

And solving the Km by using a double reciprocal mapping method according to the diluted concentration and the light absorption value.

The equation of mie:

double reciprocal mapping method:

wherein v is the enzyme reaction rate; [ S ]]Is the substrate concentration; v. of_maxIs the reaction rate at which the enzyme is saturated with substrate.

The results are shown in FIG. 2: polysorbate 80 group: km-0.0403, Vmax-0.8868; control group: km is 0.0147 and Vmax is 0.8300. From the results, it can be seen that the addition of polysorbate 80 competitively inhibits the degradation of naphthyl acetate by esterase, resulting in an increase in the Km.

Experiment two, detection of degradation of PS80 by HCP

The supernatant harvested after the cell culture of the CHO cells was subjected to capto blue (high sub) affinity chromatography to obtain an intermediate, from which a sample was prepared as a reaction product solution sample containing 0.04% polysorbate 80, and then incubated at 25 ℃. Polysorbate 80 concentration measurements before and after incubation are shown in table 5.

The polysorbate detection method comprises the following steps: because the concentration added in the biological medicine preparation is higher than the CMC (chemical component production and control) value, the biological medicine preparation generally exists in the form of micelle, N-phenyl-1-naphthylamine (NPN, a fluorescent dye) is combined with the micelle to generate a fluorescent signal, and the concentration of polysorbate 80 is determined by adopting a fluorescence detector for detection and external standard method quantification.

Table 5: the result of the detection

Sample information	Polysorbate 80 content% (w/v)
		Before incubation	0.039
After incubation	0.022

In this example, experiment one uses a competitive inhibition method to prove that the same esterase is used for degradation of naphthyl ester and degradation of polysorbate, and experiment two also detects degradation of polysorbate by chromatography. The two experiments show two methods, namely directly detecting the degradation activity of the polysorbate by esterase and the correlation of the degradation activity of the esterase to naphthyl ester, and further complementally show that the degradation activity of the enzyme to the polysorbate can be represented by using the naphthyl ester as an enzyme activity characterization agent. Because the degradation rate of the polysorbate is slow, the degradation pathways are diversified, and the degradation is greatly influenced by other physical and chemical factors, the direct detection of the degradation of the polysorbate wastes time and labor and is easily interfered. By the method, namely the method for representing the esterase activity by naphthyl ester, the degradation of the polysorbate by HCP esterase in the product is predicted, the cost is low, the operation is easy, and the method is extremely easy to use for the research of the purification process in the product development and the risk evaluation of the degradation activity of the polysorbate by the esterase in the product.

Example 4: detection of esterase specific activity in intermediate product

Standard curve

A standard curve was made as described in example 1.

Activity detection

In this example, the intermediate product sample is an intermediate product solution obtained by subjecting a supernatant obtained by culturing CHO cells to cell culture to capto blue (high sub) affinity chromatography. HCP concentration in the sample was 1.0X 10³ppm。

The substrate solution was prepared as 0.04mol/L disodium hydrogenphosphate/sodium dihydrogenphosphate buffer pH 7.0, 1% (v/v) acetone, 0.0003mol/L alpha-naphthyl acetate. A reaction mixture (600. mu.L) was prepared in duplicate by mixing 100. mu.L of the sample with 500. mu.L of the prepared substrate solution. A portion of the reaction mixture was taken as a substrate for color development reaction and immediately subjected to color development reaction and absorbance was measured and recorded as "at 0" value. The other part of the reaction mixture was incubated at 30 ℃ for 1 hour to obtain a reaction product solution, and then a color development reaction was carried out using the reaction product solution obtained by incubation as a color development reaction substrate and absorbance was measured.

table 6: the result of the detection

Absorbance of the solution	At 0 time	1 hour
			600nm	3.49	4.69

The volume activity of the esterase in the intermediate product of this example was calculated to be 0.12U/mL product solution and the specific activity was calculated to be 0.125U/mg HCP esterase protein.

Example 5: detection of esterase specific activity in intermediate product

The intermediate samples, solutions were prepared and the experimental procedure was the same as in example 4, except that the incubation time of the substrate solution with the sample solution was 2 hours. The detection results are as follows:

table 7: the result of the detection

Absorbance of the solution	At 0 time	2 hours
			600nm	3.49	5.67

Calculated, volume viability: 0.12U/mL product solution; the specific activity is as follows: 0.122U/mg HCP esterase protein.

Example 6: detection of esterase specific activity in intermediate product

The intermediate samples, solutions were prepared and the experimental procedure was the same as in example 4, except that the incubation time of the substrate solution with the sample solution was 3 hours. The detection results are as follows:

table 8: the result of the detection

Absorbance of the solution	At 0 time	3 hours
			600nm	3.49	6.13

Calculated, volume viability: 0.10U/mL of product solution; the specific activity is as follows: 0.100U/mg HCP esterase protein.

Example 7: detection of specific activity of esterase in intermediate product

The intermediate samples, solutions were prepared and the experimental procedure was the same as in example 4, except that the incubation time of the substrate solution with the sample solution was 6 hours. The detection results are as follows:

table 9: the result of the detection

Absorbance of the solution	At 0 time	6 hours
			600nm	3.49	7.83

Calculated, volume viability: 0.08U/mL product solution; the specific activity is as follows: 0.085U/mg HCP esterase protein.

Example 8: detection of esterase specific activity in end product

Standard curve

A standard curve was made as described in example 1.

Activity detection

In this example, the final product sample was obtained from the finished product obtained from CHO-produced monoclonal antibody. The HCP concentration in the sample was 10.4 ppm.

The substrate solution was prepared as 0.04mol/L disodium hydrogenphosphate/sodium dihydrogenphosphate buffer pH 7.0, 1% (v/v) acetone, 0.0003mol/L alpha-naphthyl acetate. Duplicate reaction mixtures (600. mu.L) were prepared by mixing 100. mu.L of the final solution with 500. mu.L of the prepared substrate solution. A portion of the reaction mixture was taken as a substrate for color development reaction and immediately subjected to color development reaction and absorbance was measured and recorded as "at 0" value. The other part of the reaction mixture was incubated at 30 ℃ for 24 hours to obtain a reaction product solution, and then a color development reaction was carried out using the reaction product solution obtained by incubation as a color development reaction substrate and absorbance was measured.

table 10: the result of the detection

Absorbance of the solution	At 0 time	24 hours
			600nm	0.78	2.39

Calculated volume viability: 0.01U/mL of product solution; the specific activity is as follows: 0.700U/mg HCP esterase protein.

Example 9: detection of esterase specific activity in end product

The final product samples, solutions were prepared and the experimental procedure was the same as in example 8, except that the incubation time of the substrate solution with the sample solution was 72 hours. The detection results are as follows:

table 11: the result of the detection

Absorbance of the solution	At 0 time	72 hours
			600nm	0.78	3.91

Calculated volume viability: 0.01U/mL of product solution; the specific activity is as follows: 0.480U/mg HCP esterase protein.

Based on the above embodiments, in combination with the whole technical information in this document, the parameters such as materials, steps, conditions, numerical values or numerical value ranges can be increased, decreased and changed according to the actual conditions and requirements without significantly affecting the implementation of the effects of the present invention, which is easily conceived and done by those skilled in the art, and the embodiments obtained by such increase, decrease and change also belong to the scope of the present invention protected by the present application.

Claims

1. A method of characterizing polysorbate degrading activity of an esterase in a host cell protein, the method comprising:

a) reacting a sample comprising host cell proteins with naphthyl acetate;

2. The method of claim 1, wherein the naphthyl acetate is alpha-naphthyl acetate and the absorbance is detected at 600 nm; alternatively, the naphthyl acetate is β -naphthyl acetate, and the absorbance is measured at 550 nm.

3. The method according to claim 1, wherein in the step a), naphthyl acetate is contained in a substrate solution to react with the sample, and the substrate solution contains 0.01 to 0.2mol/L disodium hydrogen phosphate/sodium dihydrogen phosphate buffer, pH 6.0 to 8.0, 0.5 to 3% (v/v) acetone and 0.00001 to 0.006mol/L naphthyl acetate.

4. The method of claim 1, wherein the reacting of the sample with naphthyl acetate comprises incubating at 25-30 ℃ for 0.5-120 hours; preferably, when the sample is an intermediate product after host cell culture and harvest, incubating for 0.5-6 hours; preferably, when the sample is the final product, the incubation is carried out for 6 to 120 hours.

5. The method according to claim 1, wherein in step B), the fast blue B salt is contained in a developing solution further comprising sodium dodecyl sulfate; preferably, the color development liquid is a mixture of 5% (w/v) sodium dodecyl sulfate solution and 1% (w/v) fast blue B salt solution according to a volume ratio of 10: 1-1: 1.

6. The method according to claim 1, wherein the color reaction in step b) comprises centrifuging the color reaction system at 4000-10000 rpm for 5-10 minutes; the supernatant from the centrifugation was then assayed for absorbance as described in step c).

7. The method according to claim 3, wherein the volume ratio of the sample to the substrate solution is 1:1 to 1: 10.

8. The method according to claim 5, wherein the volume ratio of the developing solution to the sample in step a) is 1: 1.

9. The method of claim 1, further comprising determining from the measured absorbance the enzymatic activity, specific activity and/or unit volume activity of the esterase in the sample to quantitatively characterize the degradation activity of the esterase on polysorbates in host cell proteins.

10. The method of claim 1, further having one or more of the following features:

the sample is an intermediate product or a final product after culture and harvest of the host cell;

the host cell is a mammalian cell, such as a CHO cell;

the recombinant protein is an antibody; and/or

The polysorbate is selected from polysorbate 80 and polysorbate 20.