CN114814000A - Method for detecting content of recombinant human interferon - Google Patents
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Abstract
The invention relates to a method for detecting the content of recombinant human interferon, which comprises the following steps: constructing a peak area-concentration standard curve of the recombinant human interferon; carrying out affinity high performance liquid chromatography detection on a sample to be detected, and substituting a detection result into a peak area-concentration standard curve of the recombinant human interferon to calculate to obtain the content of the recombinant human interferon in the sample to be detected; the chromatographic column adopted by the affinity high performance liquid chromatography detection comprises a filler, and the filler is agarose gel coupled with a recombinant human interferon monoclonal antibody. The method for detecting the content of the recombinant human interferon can realize effective separation of impurities and accurate detection of the content of the recombinant human interferon in a sample to be detected. Meanwhile, the content detection method can detect the recombinant human interferon sample with higher purity and the recombinant human interferon sample with complex components, and has low quantitative limit.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for detecting the content of recombinant human interferon.
Background
The recombinant human interferon has wide antiviral effect, mainly induces various antiviral proteins by combining the interferon with a target cell surface interferon receptor, prevents the synthesis of viral proteins, inhibits the replication and transcription of viral nucleic acid, has multiple immunoregulation functions, can improve the phagocytic activity of macrophages, and promotes and maintains the functions of immune monitoring, immune protection and immune homeostasis of an organism.
The research on the recombinant human interferon by people has been up to decades, and the method for detecting the content of the recombinant human interferon is also continuously innovated. At present, for the content detection of the recombinant human interferon alpha 1b, the commonly used methods are mainly divided into two types according to the detection purpose: one is the method for detecting the content of interferon with biological activity, including the plaque reduction method, the cytopathic inhibition test and the virus quantitative method, and the other is the method for detecting the chemical content of interferon, including the ELISA method, the lowry method, the direct quantitative method of a micro ultraviolet spectrophotometer, etc. The ELISA method can be used for detecting a recombinant human interferon sample with high purity and a recombinant human interferon sample with complex components and low purity due to the specificity, such as a bacterial strain breaking supernatant sample after fermentation of recombinant human interferon alpha 1b engineering bacteria, wherein the recombinant human interferon alpha 1b engineering bacteria contain a large amount of hybrid protein, nucleic acid, polysaccharide and the like, and only a small part of the recombinant human interferon alpha 1b is detected. The principle of ELISA method for detecting human interferon content is as follows: the human interferon monoclonal antibody is coated on the micropore reaction plate, the interferon in the sample to be detected and the standard substance can be combined with the human interferon monoclonal antibody, and free components are washed away; then adding horseradish peroxidase labeled secondary antibody to combine with interferon, and washing free components; adding color-developing agent, if there is interferon in the reaction hole, horseradish peroxidase can make colorless color-developing agent appear blue, adding stopping liquid to turn yellow, and judging the existence or quantity of interferon according to the color-developing depth. However, in practical detection, the content detection of the recombinant human interferon alpha 1b in a bacteria-breaking supernatant sample after fermentation by an ELISA method has poor repeatability and unstable recovery rate, so that the reference value of detection data is low.
In addition, the method also provides a method for quantitatively detecting the content of the intermediate of the recombinant human alpha interferon process by affinity chromatography. The method comprises the following steps: the construction of engineering bacteria for expressing recombinant human alpha interferon, fermenting the engineering bacteria to obtain target protein expressed in the form of soluble protein or inclusion body, then carrying out coarse purification and DEAE anion exchange column chromatography to obtain an intermediate, and then quantitatively detecting the content of the recombinant human alpha interferon in the intermediate by using a monoclonal antibody gel affinity chromatography column. The method is characterized in that: a sample quantitatively detected by a monoclonal antibody gel layer chromatographic column is an intermediate, and the purity of the sample is higher through crude purification and DEAE anion exchange column chromatography; secondly, the used monoclonal antibody gel chromatography column is a preparation-grade chromatography column, and the used equipment is a preparation-grade purifier, so that the sample demand is large during detection, and the detection time of a single sample is long; and thirdly, the content detection range of the recombinant human alpha interferon is 0.5-30 mg/mL. Therefore, the method has requirements on the purity of the detected sample, is only suitable for the high-purity sample subjected to primary purification, has large sample demand and high content detection range, and is not suitable for the sample with complex detected components and low content.
Disclosure of Invention
Based on the above, the invention provides the method for detecting the content of the recombinant human interferon, which has the advantages of small sample demand, suitability for detecting samples with complex components and low recombinant human interferon content (such as a sample of a bacterium breaking supernatant after fermentation of recombinant human interferon alpha 1b engineering bacteria) and accurate detection result.
The specific technical scheme is as follows:
a method for detecting the content of recombinant human interferon comprises the following steps:
preparing recombinant human interferon reference substance solutions with different concentrations;
performing affinity high performance liquid chromatography detection on the recombinant human interferon reference substance solutions with different concentrations to construct a peak area-concentration standard curve of the recombinant human interferon;
carrying out affinity high performance liquid chromatography detection on a sample to be detected, substituting a detection result into the peak area-concentration standard curve of the recombinant human interferon to calculate, and obtaining the content of the recombinant human interferon in the sample to be detected;
wherein, the chromatographic column adopted by the affinity high performance liquid chromatography detection comprises a filler, and the filler is sepharose gel coupled with a recombinant human interferon monoclonal antibody;
the elution procedure adopted by the affinity high performance liquid chromatography detection comprises the following steps:
(1) balancing the chromatographic column with a balancing solution, wherein the balancing solution comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride;
(2) eluting the chromatographic column for the first time by using a first eluent, wherein the first eluent comprises the components of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride and tween 20;
(3) equilibrating the column with the equilibration solution;
(4) eluting the column a second time with a second eluent having a composition comprising glycine.
In one embodiment, in the first eluent, the concentration of the disodium hydrogen phosphate dodecahydrate is 5.4-6.6 g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-1.6 g/L, the concentration of the sodium chloride is 57-60 g/L, and the concentration of the Tween 20 is 4-6 mL/L.
In one embodiment, the first eluent has a pH of 6.5. + -. 0.2 and a conductivity of 82.5. + -. 3 mS/cm.
In one embodiment, the concentration of glycine in the second eluent is 1-1.6 g/L.
In one embodiment, the pH of the second eluent is 2.5. + -. 0.2 and the conductivity is 2.4. + -. 0.5 mS/cm.
In one embodiment, the concentration of the disodium hydrogen phosphate dodecahydrate in the equilibrium liquid is 5.4-6.6 g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-1.6 g/L, and the concentration of the sodium chloride is 7-8 g/L;
and/or the pH value of the equilibrium liquid is 7.0 +/-0.2, and the conductivity is 15 +/-2 mS/cm.
In one embodiment, the elution procedure used in the affinity high performance liquid chromatography assay comprises:
balancing the chromatographic column with the balance liquid for 0-2 min;
switching the solution for 2-3 min;
3-10 min, and carrying out first elution on the chromatographic column by using the first eluent;
switching the solution for 10-11 min;
balancing the chromatographic column with the balance liquid for 11-25 min;
switching the solution for 25-26 min;
and (8) carrying out secondary elution on the chromatographic column by using the second eluent for 26-35 min.
In one embodiment, the conditions for the affinity high performance liquid chromatography detection further comprise: the flow rate is 0.7-0.9 mL/min, the temperature is 23-27 ℃, and the wavelength of the ultraviolet detector is 275-285 nm;
and/or the sample to be detected is a fermented bacteria-breaking supernatant sample subjected to centrifugal treatment; the conditions of the centrifugation include: the rotating speed is 11500 rpm-12500 rpm, and the time is 2 min-10 min.
In one embodiment, the limit of quantitation of the content detection method is greater than or equal to 20 μ g/mL.
In one embodiment, the recombinant human interferon is IFN α 1b, IFN α 2b or IFN α 2 a.
The method for detecting the content of the recombinant human interferon adopts agarose gel coupled with the recombinant human interferon monoclonal antibody as chromatographic column packing, combines a proper elution program, can realize effective separation of impurities and accurate detection of the content of the recombinant human interferon in a sample to be detected, and is suitable for detecting parameters such as fermentation expression quantity of the recombinant human interferon.
Meanwhile, the content detection method can detect the recombinant human interferon sample with higher purity and the recombinant human interferon sample with complex components, such as the content of the recombinant human interferon in a bacteria-breaking supernatant sample, has stable recovery rate and good repeatability, has short detection time and low detection sample demand, is calculated by 0.5mL, only needs 10 mu g of recombinant human interferon at minimum, has low limit of quantitation (more than or equal to 20 mu g/mL), and has great advantage for detecting the content of the recombinant human interferon in a sample to be detected by shake flask fermentation.
Drawings
FIG. 1 is a chromatogram of blank solution-1 during the standard curve establishment in example 1;
FIG. 2 is a chromatogram of blank solution-2 during the standard curve establishment in example 1;
FIG. 3 is a chromatogram of a physicochemical control 20 μ g/mL human interferon α 1b sample obtained in the process of establishing a standard curve in example 1;
FIG. 4 is a chromatogram of a physicochemical control 40 μ g/mL human interferon α 1b sample obtained in the process of establishing a standard curve in example 1;
FIG. 5 is the chromatogram of the physicochemical control of 80. mu.g/mL human interferon alpha 1b in the process of establishing the standard curve in example 1;
FIG. 6 is a chromatogram of a physicochemical reference substance of 120. mu.g/mL human interferon alpha 1b in the process of establishing a standard curve in example 1;
FIG. 7 is the chromatogram of the physicochemical control 160. mu.g/mL human interferon alpha 1b in the process of establishing the standard curve in example 1;
FIG. 8 is a chromatogram of a physicochemical control sample of human interferon alpha 1b at 200. mu.g/mL in the process of establishing a standard curve in example 1;
FIG. 9 is a detection chromatogram of sample a in example 1;
FIG. 10 is a detection chromatogram of sample b in example 1;
FIG. 11 is a detection chromatogram of sample c in example 1;
FIG. 12 is a chromatogram of a blank solution-1 during the establishment of a standard curve in example 1;
FIG. 13 is a graph showing the comparison of the detection results of 9 batches of shake flask fermentation broken bacteria supernatant samples respectively detected by the content detection method of example 1 and the traditional ELISA method (the abscissa is the batch number of the sample, and the ordinate is the concentration of recombinant human interferon alpha 1b in the sample).
Detailed Description
The method for detecting the recombinant human interferon content of the present invention is further described in detail with reference to the following specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor are they to imply that the importance or quantity of the technical feature being indicated is implicitly specified. Also, "first," "second," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as constituting a closed limitation to the number.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical intervals are regarded as continuous, and include the minimum and maximum values of the range and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid mixing, and volume percentages for liquid-liquid mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
In the present invention, the solvent used in each solution is water unless otherwise specified.
In the present invention, "affinity high performance liquid chromatography" refers to immunoaffinity chromatography, which is a method for quantitatively analyzing an antigen or an antibody by using a high specific affinity between the antigen and the antibody.
In the invention, the expression quantity of recombinant human interferon fermentation refers to the content of recombinant human interferon contained in each milliliter of bacterial liquid after the fermentation of the genetic engineering bacteria.
The invention provides a method for detecting the content of recombinant human interferon, which comprises the following steps:
preparing recombinant human interferon reference substance solutions with different concentrations;
performing affinity high performance liquid chromatography detection on the recombinant human interferon reference substance solutions with different concentrations to construct a peak area-concentration standard curve of the recombinant human interferon;
carrying out affinity high performance liquid chromatography detection on a sample to be detected, substituting a detection result into the peak area-concentration standard curve of the recombinant human interferon to calculate, and obtaining the content of the recombinant human interferon in the sample to be detected;
wherein, the chromatographic column adopted by the affinity high performance liquid chromatography detection comprises a filler, and the filler is sepharose gel coupled with a recombinant human interferon monoclonal antibody;
the elution procedure adopted by the affinity high performance liquid chromatography detection comprises the following steps:
(1) balancing the chromatographic column with a balancing solution, wherein the balancing solution comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride;
(2) eluting the chromatographic column for the first time by using a first eluent, wherein the first eluent comprises the components of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride and tween 20;
(3) equilibrating the column with the equilibration solution;
(4) eluting the column a second time with a second eluent having a composition comprising glycine.
In one example, the elution procedure further comprises:
(5) washing the chromatographic column with a washing solution;
(6) equilibrating the column with an equilibration solution.
It will be appreciated that step (5) is aimed at better preservation of the column to extend its lifetime and reduce assay costs and step (6) is aimed at preparing for the next assay.
Without limitation, the detection principle of the method for detecting the content of the recombinant human interferon is as follows: the agarose gel coupled with the recombinant human interferon monoclonal antibody is used as chromatographic column packing, the recombinant human interferon in a sample to be detected can be subjected to specific affinity adsorption, then impurities are removed by using a specific elution program, the recombinant human interferon is desorbed, a map is obtained by detection, the peak area of the recombinant human interferon in the map is calculated, and the content of the recombinant human interferon in the sample to be detected is calculated through a standard curve established by reference substance concentration and the peak area.
In one example, the retention time of the recombinant human interferon peak is 29.3 ± 0.6 min.
In one example, the integration condition for the map is peak width 50, threshold 20. Optionally, the integration interval is 25min to 40 min.
It is understood that, according to the type of recombinant human interferon to be detected, the selection of the recombinant human interferon monoclonal antibody is different, and the two are mutually adaptive. Specifically, the recombinant human interferon species can be IFN alpha 1b, IFN alpha 2a, and the like.
Without limitation, the sample to be detected in the method for detecting the content of recombinant human interferon may be a purified sample with high purity of recombinant human interferon, or a sample with complex components and low purity. Without limitation, when the concentration of the recombinant human interferon in the sample to be detected is higher, the sample can be properly diluted, and when the concentration is lower, the sample is not diluted. The concentration of the recombinant human interferon in the sample to be detected can be preferably 80-120 mu g/mL (estimated value).
Particularly, the method for detecting the content of the recombinant human interferon is suitable for samples with complex components and low purity, such as fermented bacteria-breaking supernatant samples. When the content of the sample to be detected is detected, proper pretreatment can be carried out. In one example, the fermented broken bacteria supernatant sample is centrifuged to be used as a sample to be tested, and the centrifugation conditions comprise: the rotating speed is 11500 rpm-12500 rpm, and the time is 2 min-10 min. Further, the method also comprises a step of filtering with a 0.22 μm filter membrane. The sample processing method is simple, and can process a plurality of samples simultaneously.
In one example, the agarose gel is cyanogen bromide activated agarose gel. Specifically, the agarose gel is CNBr Activated Sepharose 4 FF.
In one example, the preparation method of the filler comprises the following steps:
(1) obtaining cell culture supernatant containing human interferon alpha 1b antibody, concentrating, affinity chromatography to purify human interferon alpha 1b antibody;
(2) coupling the purified human interferon alpha 1b antibody with agarose gel, comprising the steps of:
mixing the purified human interferon alpha 1b antibody with agarose gel, mixing the obtained mixture at room temperature for 1-10 h or standing overnight (such as 10-20 h) at 0-5 ℃, sealing and washing.
In one example, the concentrations of the recombinant human interferon control solutions with different concentrations are 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 120 μ g/mL, 160 μ g/mL and 200 μ g/mL respectively, and the solvent is the equilibrium solution.
In one example, the limit of quantitation of the content detection method is greater than or equal to 20. mu.g/mL. Furthermore, the limit of quantitation of the content detection method is 20 mu g/mL-30 mg/mL.
In one example, in the first eluent, the concentration of the disodium hydrogen phosphate dodecahydrate is 5.4-6.6 g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-1.6 g/L, the concentration of the sodium chloride is 57-60 g/L, and the concentration of the Tween 20 is 4-6 mL/L. Specifically, in the first eluent, the concentration of the disodium hydrogen phosphate dodecahydrate is 5.95g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1.31g/L, the concentration of the sodium chloride is 58.44g/L, and the concentration of the Tween 20 is 5 mL/L.
In one example, the first eluent has a pH of 6.5. + -. 0.2 and a conductivity of 82.5. + -.3 mS/cm.
In one example, the first eluate is filtered through a 0.22 μm filter before being subjected to the elution procedure.
In one example, the concentration of the glycine in the second eluent is 1-1.6 g/L. Specifically, the concentration of the glycine in the second eluent is 1.5 g/L.
In one example, the second eluent has a pH of 2.5. + -. 0.2 and a conductivity of 2.4. + -. 0.5 mS/cm. In particular, hydrochloric acid may be used to adjust the pH.
In one example, the second eluate is filtered through a 0.22 μm filter before being subjected to the elution procedure.
In one example, the concentration of the disodium hydrogen phosphate dodecahydrate is 5.4-6.6 g/L, the concentration of the sodium dihydrogen phosphate dihydrate is 1-1.6 g/L, and the concentration of the sodium chloride is 7-8 g/L in the equilibrium liquid. Specifically, in the equilibrium solution, the concentration of disodium hydrogen phosphate dodecahydrate is 5.95g/L, the concentration of sodium dihydrogen phosphate dihydrate is 1.31g/L, and the concentration of sodium chloride is 7.31 g/L.
In one example, the pH value of the equilibrium liquid is 7.0 +/-0.2, and the conductivity is 15 +/-2 mS/cm.
In one example, the equilibration solution is filtered through a 0.22 μm filter before being subjected to the elution procedure.
In one example, the cleaning solution comprises acetic acid and sodium chloride.
In one example, the cleaning solution includes: acetic acid with a concentration of 4-8 mL/L and sodium chloride with a concentration of 28-32 g/L. Specifically, the cleaning liquid includes: acetic acid at a concentration of 6mL/L and sodium chloride at a concentration of 29.22 g/L.
In one example, the pH value of the cleaning solution is 4.0 +/-0.2, and the conductivity is 45 +/-2 mS/cm. In particular, the pH can be adjusted with sodium hydroxide.
In one example, the wash solution is filtered through a 0.22 μm filter before the elution process is performed.
In one example, the elution procedure used in the affinity high performance liquid chromatography assay comprises:
balancing the chromatographic column with the balance liquid for 0-2 min;
switching the solution for 2-3 min;
3-10 min, and carrying out first elution on the chromatographic column by using the first eluent;
switching the solution for 10-11 min;
balancing the chromatographic column with the balance liquid for 11-25 min;
switching the solution for 25-26 min;
and (8) carrying out secondary elution on the chromatographic column by using the second eluent for 26-35 min.
In one example, the elution procedure used in the affinity high performance liquid chromatography assay comprises:
balancing the chromatographic column with the balance liquid for 0-2 min;
switching the solution for 2-3 min;
3-10 min, and carrying out first elution on the chromatographic column by using the first eluent;
switching the solution for 10-11 min;
balancing the chromatographic column with the balance liquid for 11-25 min;
switching the solution for 25-26 min;
performing a second elution on the chromatographic column with the second eluent for 26-35 min;
switching the solution for 35-36 min;
washing the chromatographic column with the washing liquid for 36-40 min;
switching the solution for 40-41 min;
and balancing the chromatographic column by using the balance liquid for 41-55 min.
In one example, a blank solution detection step is further included in the process of constructing a peak area-concentration standard curve of the recombinant human interferon.
In one example, the blank solution comprises: tris with a concentration of 9.5-10.5 mM, EDTA with a concentration of 0.5-1.5 mM, and sodium chloride with a concentration of 0.05-0.15 mM. Specifically, the blank solution comprises: tris at a concentration of 10mM, EDTA at a concentration of 1mM and sodium chloride at a concentration of 0.1 mM. Further, lysozyme is added before the blank solution is used, wherein the concentration of the lysozyme is 0.5-1.5 mg/mL.
In one example, the blank solution has a pH of 8.0 ± 0.2. In particular, hydrochloric acid may be used to adjust the pH.
In one example, the pH value of the cleaning solution is 4.0 +/-0.2, and the conductivity is 45 +/-2 mS/cm. In particular, the pH can be adjusted with sodium hydroxide.
In addition, in one example, the conditions for the affinity high performance liquid chromatography detection further include: the flow rate is 0.7-0.9 mL/min, the temperature is 23-27 ℃, and the wavelength of the ultraviolet detector is 275-285 nm.
Specific examples are as follows.
The high performance liquid analytical column used in the examples is filled with self-made monoclonal antibody agarose gel, the specification is 4.6mm x 50mm, and the total column plate number of the column effect is more than or equal to 100.
The main preparation steps of the monoclonal antibody agarose gel adopted in the embodiment are as follows: (1) culturing the hybridoma 22B8-D5-E8-3C4 to obtain a cell culture supernatant containing the human interferon alpha 1B antibody; (2) filtering and concentrating cell culture supernatant, and performing affinity chromatography by using a chromatographic column filled with MabSelect Sure filler to obtain a pure human interferon alpha 1b antibody, wherein specifically, the 2-step affinity chromatography comprises the following steps: the mobile phase for affinity chromatography in step 1 comprises equilibrium solution, 0.02M Tris-HCl with pH7.5 and 1.5M NaCl, eluent, pH3.0.05M citric acid; the step 2 is the same as the step 1 in the affinity chromatography mobile phase; (3) coupling a human interferon alpha 1b antibody to CNBr Activated Sepharose 4FF cyanogen bromide Activated agarose gel to obtain monoclonal antibody agarose gel, wherein the coupling step comprises the following steps: firstly, taking a proper amount of CNBr Activated Sepharose 4FF cyanogen bromide Activated agarose gel dry powder, rapidly expanding in a solution a (1mM HC1, 0.5M NaCl, pH 3.0), wherein about 1 gram of the dry powder can expand to 3ml of final volume, placing the powder on a glass filtering crucible, washing the powder for 30min by using the solution a, and washing about 1 gram of the dry powder by using 200ml of the solution a; ② solution b (0.2M NaHCO) for human interferon alpha 1b antibody 3 0.5M NaCl, pH8.3) and diluted to 5mg/ml with solution b; and thirdly, mixing the washed gel and the antibody in equal volume, and uniformly mixing for 3 hours at room temperature. Fourthly, washing with 20mM Tris-HCl with pH8.3, sealing and placing for 3 hours at room temperature; washing with 0.05M Tris-HCl solution (pH8.0) and 0.2M acetic acid buffer solution (pH3.0) for 4 times alternately, each time with 3 times of gel volume; sixthly, washing the mixture by using PBS solution with 10 times of column volume to obtain the monoclonal antibody agarose gel.
The physical and chemical reference substance of the recombinant human interferon alpha 1b adopted in the embodiment is subjected to protein concentration calibration by a Lowry method, is subpackaged to 1 ml/branch, and is stored at the temperature of minus 80 ℃.
The acquisition mode of the fermented lysozyme bacteria-breaking supernatant adopted in the embodiment is as follows: sampling fermentation liquor in the fermentation process of a 20L fermentation tank, centrifuging 1ml of fermentation liquor at 10000rpm for 5min to collect thallus, adding 1ml of lysozyme at 1mg/ml, mixing uniformly, breaking the thallus at room temperature for 1h, repeatedly freezing and thawing at minus 80 ℃ for 3 times, centrifuging at 10000rpm for 10min, and collecting supernatant.
The shake flask fermentation adopted in the comparative example breaks the clear supernatant sample and obtains the mode: sampling fermentation liquor in the 50ml shaking flask fermentation process, centrifuging 10ml fermentation liquor at 10000rpm for 5min to collect thallus, adding 1mg/ml lysozyme 1ml, mixing, breaking the thallus at room temperature for 1h, repeatedly freezing and thawing at-80 ℃ for 3 times, centrifuging at 10000rpm for 10min, and collecting supernatant.
Example 1
This example is a method for detecting recombinant human interferon content, comprising the following steps:
(1) the detection solution was prepared, and there were 5 solutions as follows:
A. balance liquid: comprises 5.95g/L disodium hydrogen phosphate dodecahydrate, 1.31g/L sodium dihydrogen phosphate dihydrate and 7.31g/L sodium chloride, has pH value of 7.0 and conductivity of 15mS/cm, and is filtered by a 0.22 mu m filter membrane for later use.
B. Washing the impurities: comprises 5.95g/L disodium hydrogen phosphate dodecahydrate, 1.31g/L sodium dihydrogen phosphate dihydrate, 58.44g/L sodium chloride and 205 mL/L Tween, has pH value of 6.5 and conductivity of 82.5mS/cm, and is filtered through a 0.22 mu m filter membrane for standby.
C. Eluent: contains 1.5g/L glycine, is adjusted to pH 2.5 with 6M hydrochloric acid, has a conductivity of 2.4mS/cm and is filtered through a 0.22 μ M filter for further use.
D. Cleaning solution: containing acetic acid 6ml/L and NaCl 29.22g/L, adjusted to pH 4.0 with 1M NaOH, conductivity 45mS/cm and filtered through a 0.22 μ M filter for further use.
E. Blank solution: containing Tris 10mM, EDTA 1mM and NaCl 0.1M, adjusted to pH8.0 with HCl, lysozyme was added at a concentration of 1mg/mL before use.
(2) The sample detection is carried out by adopting a high performance liquid chromatography system, the flow rate is 0.8mL/min, the temperature is 25 ℃, the wavelength of an ultraviolet detector is 280nm, the sample injection volume is 100 mu L, and the elution gradient of the detection method is shown in the following table 1:
TABLE 1
| Time (min) | A(％) | B(％) | C(％) | D(％) |
| 0 | 100 | 0 | 0 | 0 |
| 2 | 100 | 0 | 0 | 0 |
| 3 | 0 | 100 | 0 | 0 |
| 10 | 0 | 100 | 0 | 0 |
| 11 | 100 | 0 | 0 | 0 |
| 25 | 100 | 0 | 0 | 0 |
| 26 | 0 | 0 | 100 | 0 |
| 35 | 0 | 0 | 100 | 0 |
| 36 | 0 | 0 | 0 | 100 |
| 40 | 0 | 0 | 0 | 100 |
| 41 | 100 | 0 | 0 | 0 |
| 55 | 100 | 0 | 0 | 0 |
(3) The integral conditions of the high performance liquid chromatography ultraviolet spectrum are that the peak width is 50, the threshold value is 20, the integral interval is 25 min-40 min, and the target protein peak retention time is 29.3 min.
(4) Establishing a standard curve: firstly, taking 1mL of blank solution, adopting the method in the step (2) to sample 2 needles, carrying out peak area integration on an ultraviolet absorption peak with the retention time of about 29.3min, and taking an average value S of the areas of 2 times of peaks; and (2) taking a physicochemical reference substance of human interferon alpha 1b, diluting the reference substance by the balance liquid according to the ratio of 20 mu g/mL, 40 mu g/mL, 80 mu g/mL, 120 mu g/mL, 160 mu g/mL and 200 mu g/mL to prepare 6 samples, wherein each sample is about 1mL, sampling for 1 time by adopting the method in the step (2), carrying out peak area integration on ultraviolet absorption peaks with the retention time of about 29.3min, subtracting the noise peak area S, then establishing a standard curve by taking the peak area as a vertical coordinate and the reference substance concentration as a horizontal coordinate, and fitting the standard curve with the goodness R2 of not less than 0.995.
(5) Sample treatment and detection: taking three batches of fermented lysozyme bacteria-breaking supernatant samples, respectively recording the samples as a sample a, a sample b and a sample c, firstly centrifuging at 12000rpm for 5min, then filtering by using a 0.22 mu m needle filter membrane, injecting 100 mu L of sample, and subtracting a noise peak area S from a peak area to obtain the content of the recombinant human interferon alpha 1b in the sample, wherein the results are shown in the following table 2 and figures 1-11. In addition, FIG. 12 is the whole chromatogram of blank solution-1, which is characterized by comprising the ultraviolet absorption peak of the sample hetero protein (containing lysozyme in the blank solution) flowing through for 0-4min, the ultraviolet absorption peak of the first eluent for 4-14min, the ultraviolet absorption peak of the recombinant human interferon alpha 1b at the position of 29.3 +/-0.6 min, and the ultraviolet absorption peak of the cleaning solution for 37-38 min.
TABLE 2
Example 2
The method for detecting the content of the embodiment 1 is comprehensively verified by methodology, and includes verification items such as system applicability, specificity, linearity, accuracy, precision, intermediate precision, detection limit and quantification limit. The content detection method has high accuracy, the recovery rate of the added standard is between 90% and 110%, and the detection precision is high, which indicates that the content detection method has good repeatability. The limit of quantitation is as low as 20 mug/mL recombinant human interferon alpha 1 b. The establishment of the content detection method ensures that the result of the expression quantity measurement of the human interferon alpha 1b in the fermentation process optimization process is more accurate and credible, and has very high guiding significance for the development of the process optimization. The following is a specific verification process.
(1) System suitability verification
The system applicability checks whether a high performance liquid chromatography system and a chromatographic column meet the detection requirements, and determines whether the detection result is accurate and reliable or not by checking the retention time (Rt) of each main characteristic peak and the Relative Standard Deviation (RSD) of the peak area, and whether the detection result is suitable for detecting the content of the recombinant human interferon alpha 1b or not.
Taking a physicochemical reference substance solution of the recombinant human interferon alpha 1b, and diluting the solution to 100 mu g/mL by using a balance solution to be used as a system applicability solution. And (4) carrying out sample injection twice on the blank solution, carrying out continuous sample injection 6 times on the system applicability solution, and recording a chromatogram. The blank solution is required to be free of interference, the retention time RSD of the main peak of the solution with the applicability of the system is less than or equal to 1.0 percent, and the area RSD of the main peak is less than or equal to 2.0 percent. The verification results are shown in table 3 below.
TABLE 3
As can be seen from the above table, a noise peak is also generated at the target peak position after the blank solution is injected, and the noise peak area needs to be deducted in the subsequent sample detection. The system applicability solution retention time RSD and the main peak area RSD both meet the requirements.
(2) Attribute validation
The specificity verification means that the ability of a detected object can be correctly determined by adopting an analysis method under the possible existence of other components (such as impurities, degradation products, auxiliary materials and the like), and for the broken bacteria supernatant of the human interferon alpha 1b fermentation liquor, the solvent is TE buffer solution which contains lysozyme, and whether the lysozyme TE solution (blank solution) interferes with the detection of the protein expression quantity needs to be verified.
Taking a physicochemical reference substance of the recombinant human interferon alpha 1b, and diluting the physicochemical reference substance to 100 mu g/mL by using a balance solution to serve as a reference substance. Collecting the fermented lysozyme supernatant as a test sample. The blank solution, the reference substance and the sample are added for 1 time respectively, and the chromatogram is recorded. The deviation between the main peak retention time of the test solution and the main peak retention time of the reference solution is required to be less than 1%, and the noise peak area of the blank solution is required to be less than 5% of the main peak area of the reference. The verification results are shown in table 4 below.
TABLE 4
| Sample name | Rt(min) | Peak area (microvolt second) |
| Blank solution | 29.826 | 4633 |
| Reference substance | 29.676 | 376482 |
| Test article | 29.743 | 230447 |
The deviation between the main peak retention time of the test sample and the main peak retention time of the reference solution is 0.2%, and the retention times are basically consistent. The area of the noise peak of the blank solution accounts for 1.2 percent of the area of the main peak of the reference substance and is less than 5 percent, and the verification result meets the requirement.
(3) Linear verification
Diluting the physicochemical reference substance of recombinant human interferon alpha 1b with balance solution at 20 μ g/mL, 40 μ g/mL, 80 μ g/mL, 120 μ g/mL, 160 μ g/mL, and 200 μ g/mL, sequentially sampling blank solution and reference substance for 2 times, detecting, taking concentration as abscissa, subtracting the average value of peak area after noise as ordinate, establishing standard curve, and requiring linear equation R 2 Not less than 0.995. The standard test data are shown in table 5 below.
TABLE 5
The data in the table are used for establishing a standard curve, and the equation is that y is 4880.1x-73193, R 2 0.9996 > 0.995, the results of the linear verification are satisfactory.
(4) Accuracy verification
Diluting three samples by using a physical and chemical reference substance of the recombinant human interferon alpha 1b according to 40 mu g/mL, 100 mu g/mL and 160 mu g/mL respectively, mixing one part of a test sample (fermented lysozyme bacteria-breaking supernatant) and a reference substance diluent according to the volume ratio of 1:1 to obtain three standard-added samples with different concentrations, injecting the three standard-added samples for three times respectively, injecting the test sample once, and calculating the concentration and the standard-added recovery rate. The average standard adding recovery rate of each concentration is required to be between 90 and 110 percent, the respective standard adding recovery rate RSD of the three samples is less than or equal to 5 percent, and the respective standard adding recovery rate RSD of the 9 samples is less than or equal to 5 percent. The results of the measurements are shown in Table 6 below.
TABLE 6
The average standard adding recovery rate under each concentration is between 90 and 110 percent, the RSD of each standard adding recovery rate of three samples is less than or equal to 5 percent, and the RSD of 9 standard adding recovery rates is less than or equal to 5 percent, thereby meeting the requirements.
(5) Precision and intermediate precision verification
The first person took the physical and chemical reference substance of the recombinant human interferon alpha 1b, diluted to 100 mug/mL with the balance solution, and prepared 6 parts in parallel as the test solution. Sampling the sample solution for 1 time, and recording the chromatogram. The retention time RSD of the main peak and the area RSD of the main peak of 6 parts of samples are required to be less than or equal to 1.0 percent and less than or equal to 5.0 percent. And the second person takes a physicochemical reference substance of the recombinant human interferon alpha 1b, dilutes the reference substance to 100 mu g/mL by using a balance solution, prepares 6 parts in parallel as a test solution, replaces one device, samples each part for 1 time, and records a chromatogram. Requiring that the peak area RSD of the main peak of the interferon in 6 test sample solutions of the second person is less than or equal to 5.0 percent; in 12 test solutions of two persons, the peak area RSD of the main peak of interferon is less than or equal to 5.0 percent. The results of the measurements are shown in Table 7 below.
TABLE 7
As shown in the table, the retention time RSD of the main peak of 6 test samples of the first person is less than or equal to 1.0 percent, and the area RSD of the main peak is less than or equal to 5.0 percent; in 6 test solutions of the second person, the peak area RSD of the main peak of the interferon is less than or equal to 5.0 percent; in 12 test solutions of two persons, the peak area RSD of the main peak of interferon is less than or equal to 5.0 percent, and the precision and the intermediate precision are qualified.
(6) Detection limit and quantitation limit verification
Taking a physicochemical reference substance of the recombinant human interferon alpha 1b, diluting with a balance solution according to 10 mu g/ml and 20 mu g/ml, and respectively taking the diluted balance substance as a detection limit sample and a quantification limit sample. And (3) taking blank solution and injecting sample twice, detecting limited sample and injecting sample 2 times, and quantifying limited sample and injecting sample 6 times. The ratio (signal-to-noise ratio) of the peak area mean value of the detection limit sample to the noise peak area mean value is required to be more than or equal to 3, the ratio of the peak area mean value of the quantification limit sample to the noise peak area mean value is required to be more than or equal to 5, and the peak area RSD of the quantification limit sample is required to be less than or equal to 10%. The results are shown in Table 8 below:
TABLE 8
As shown in the table above, the peak area signal-to-noise ratio of the detection limit sample is 5.3 > 3; the quantitative limit sample peak area signal-to-noise ratio is 7.0 more than 5, and the peak area RSD is less than 10 percent, which meets the requirement.
(7) Comparison of detection results of different temperatures
The physicochemical control solution of recombinant human interferon alpha 1b was diluted to 100. mu.g/mL with the equilibrium solution, and the detection results were observed at 23 ℃, 25 ℃ and 27 ℃ respectively, as shown in Table 9 below.
TABLE 9
| Sample name | Retention time (min) | Peak area (microvolt second) | Concentration (ug/ml) |
| 100ug/ml control (23 ℃ C.) | 28.952 | 342999 | 94.1 |
| 100ug/ml control (25 ℃ C.) | 28.952 | 361824 | 98.2 |
| 100ug/ml control (27 ℃ C.) | 28.949 | 376467 | 101.4 |
As can be seen from the above table, the analytical column increases the temperature in a proper range, and the recovery rate of human interferon alpha 1b in the sample can be increased moderately. Considering that increasing the temperature may have an effect on the lifetime of the antibodies in the analytical column, the method selects 25 ℃ (room temperature) for detection within an acceptable range of recovery.
(8) Comparison of detection results at different flow rates
The physicochemical control solution of recombinant human interferon alpha 1b was diluted to 100. mu.g/mL with the equilibrium solution, and the detection results were observed under the conditions of flow rates of 0.7mL/min, 0.8mL/min and 0.9mL/min, respectively, as shown in Table 10 below.
Watch 10
| Sample name | Retention time (min) | Peak area (microvolt second) | Concentration (ug/ml) |
| 100ug/ml reference (0.7ml/min) | 29.409 | 420854 | 111.2 |
| 100ug/ml reference (0.8ml/min) | 28.95 | 368296 | 99.6 |
| 100ug/ml reference (0.9ml/min) | 28.598 | 321941 | 89.4 |
As can be seen from the above table, the analytical column reduces the flow rate in a proper range, and the recovery rate of the recombinant human interferon alpha 1b in the sample is improved. Considering the shortening of the detection period of a single sample as much as possible, the method selects a proper flow rate of 0.8ml/min within the acceptable range of the recovery rate.
(9) Comparison of detection results at different wavelengths
Taking a physicochemical reference substance solution of the recombinant human interferon alpha 1b, diluting the solution to 100 mu g/mL by using a balance solution, detecting the solution under the conditions of 275nm, 280nm and 285nm wavelengths respectively, and observing the detection results, wherein the detection results are shown in the following table 11.
TABLE 11
| Sample name | Retention time (min) | Peak area (microvolt second) | Concentration (ug/ml) |
| 100ug/ml reference substance (275nm) | 28.953 | 354454 | 96.6 |
| 100ug/ml reference substance (280nm) | 28.955 | 363431 | 98.6 |
| 100ug/ml reference substance (285nm) | 28.951 | 310505 | 86.9 |
As can be seen from the above table, the detection result is the highest when the ultraviolet wavelength is 280nm, and the method selects the ultraviolet wavelength of 280nm within the acceptable range of the recovery rate.
Comparative example 1
And taking 9 batches of shake flask fermentation broken strain supernatant samples, and respectively adopting the content detection method of the embodiment 1 and the traditional ELISA method for detection.
The traditional ELISA method mainly comprises the following operation steps: coating an enzyme label plate with a human interferon alpha 1b monoclonal antibody according to 200 ng/hole, standing overnight at 4 ℃, and drying; washing the plate with PBST (PBS and 0.1% Tween 20), adding a blocking solution (PBST containing 2% BSA), keeping the temperature at 37 ℃ for 1h at 150 ul/hole; thirdly, adding a human interferon alpha 1b standard product, a reference product and a sample to be detected after washing the plate, keeping the temperature at 37 ℃ for 1h at 100 ul/hole; fourthly, washing the plate, adding an interferon enzyme-labeled antibody diluted by 1: 1000, keeping the temperature at 37 ℃ for 1h at 100 ul/hole; washing the plate, adding substrate (0.045% hydrogen peroxide and 0.4mg/ml o-phenylenediamine) capable of reacting with enzyme for color development, keeping the temperature at 37 ℃ away from light for 7min to ensure that the color development degree is moderate; sixthly, adding 2M sulfuric acid into 50 ul/hole to terminate the reaction, measuring the absorbance at 450nm, and printing data; and creating a standard curve according to the detection data of the standard product, and calculating the concentration of the human interferon alpha 1b in the sample to be detected according to the absorbance value of the sample to be detected.
The results are shown in FIG. 13.
FIG. 13 shows that the detection result of the content detection method of example 1 is 1.9-6.6 times of that of ELISA, which indicates that the recovery rate of human interferon alpha 1b in complex samples is low by ELISA, and the content of human interferon alpha 1b in complex samples of detection components is dominant by liquid phase method.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the present invention should be subject to the content of the appended claims, and the description and the drawings can be used for explaining the content of the claims.
Claims (10)
1. A method for detecting the content of recombinant human interferon is characterized by comprising the following steps:
preparing recombinant human interferon reference substance solutions with different concentrations;
performing affinity high performance liquid chromatography detection on the recombinant human interferon reference substance solutions with different concentrations to construct a peak area-concentration standard curve of the recombinant human interferon;
carrying out affinity high performance liquid chromatography detection on a sample to be detected, substituting a detection result into the peak area-concentration standard curve of the recombinant human interferon to calculate, and obtaining the content of the recombinant human interferon in the sample to be detected;
wherein, the chromatographic column adopted by the affinity high performance liquid chromatography detection comprises a filler, and the filler is sepharose gel coupled with a recombinant human interferon monoclonal antibody;
the elution procedure adopted by the affinity high performance liquid chromatography detection comprises the following steps:
(1) balancing the chromatographic column with a balancing solution, wherein the balancing solution comprises disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate and sodium chloride;
(2) eluting the chromatographic column for the first time by using a first eluent, wherein the first eluent comprises the components of disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride and tween 20;
(3) equilibrating the column with the equilibration solution;
(4) eluting the column a second time with a second eluent having a composition comprising glycine.
2. The method for detecting the content of recombinant human interferon according to claim 1, wherein in the first eluent, the concentration of disodium hydrogen phosphate dodecahydrate is 5.4 to 6.6g/L, the concentration of sodium dihydrogen phosphate dihydrate is 1 to 1.6g/L, the concentration of sodium chloride is 57 to 60g/L, and the concentration of Tween 20 is 4 to 6 mL/L.
3. The method for detecting the content of recombinant human interferon according to claim 1, wherein the first eluent has a pH of 6.5 ± 0.2 and a conductivity of 82.5 ± 3 mS/cm.
4. The method for detecting the content of recombinant human interferon according to claim 1, wherein the concentration of glycine in the second eluent is 1-1.6 g/L.
5. The method for detecting the content of recombinant human interferon according to claim 1, wherein the pH of the second eluent is 2.5. + -. 0.2, and the conductivity is 2.4. + -. 0.5 mS/cm.
6. The method for detecting the content of recombinant human interferon according to claim 1, wherein the concentration of disodium hydrogen phosphate dodecahydrate is 5.4 to 6.6g/L, the concentration of sodium dihydrogen phosphate dihydrate is 1 to 1.6g/L, and the concentration of sodium chloride is 7 to 8g/L in the equilibrium solution;
and/or the pH value of the equilibrium liquid is 7.0 +/-0.2, and the conductivity is 15 +/-2 mS/cm.
7. The method for detecting the content of recombinant human interferon according to claim 1, wherein the elution procedure used in the affinity high performance liquid chromatography comprises:
balancing the chromatographic column with the balance liquid for 0-2 min;
switching the solution for 2-3 min;
3-10 min, and carrying out first elution on the chromatographic column by using the first eluent;
switching the solution for 10-11 min;
balancing the chromatographic column by using the balance liquid for 11-25 min;
switching the solution for 25-26 min;
and (8) carrying out secondary elution on the chromatographic column by using the second eluent for 26-35 min.
8. The method for detecting the content of recombinant human interferon according to claim 1, wherein the conditions for the affinity high performance liquid chromatography further comprise: the flow rate is 0.7-0.9 mL/min, the temperature is 23-27 ℃, and the wavelength of the ultraviolet detector is 275-285 nm;
and/or the sample to be detected is a fermented bacteria-breaking supernatant sample subjected to centrifugal treatment; the conditions of the centrifugation include: the rotating speed is 11500 rpm-12500 rpm, and the time is 2 min-10 min.
9. The method for detecting the content of recombinant human interferon according to any one of claims 1 to 8, wherein the limit of quantitation of the method for detecting the content is not less than 20 μ g/mL.
10. The method for detecting the content of recombinant human interferon according to any one of claims 1 to 8, wherein the recombinant human interferon is IFN α 1b, IFN α 2b or IFN α 2 a.
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