CN111239278B

CN111239278B - Method for detecting recombinant human interleukin-12 protein charge variant and application

Info

Publication number: CN111239278B
Application number: CN202010080338.8A
Authority: CN
Inventors: 魏文; 王瑞玲; 刘忠凯; 王彦娜
Original assignee: Kanglitai Biomedical Qingdao Co ltd
Current assignee: Kanglitai Biomedical Qingdao Co ltd
Priority date: 2020-02-05
Filing date: 2020-02-05
Publication date: 2022-11-04
Anticipated expiration: 2040-02-05
Also published as: CN111239278A

Abstract

The invention provides a method for detecting a recombinant human interleukin-12 protein charge variant and application thereof. Analyzing a protein to be detected by a pH-IEC ion exchange chromatography to obtain a chromatogram, and determining the content of the recombinant human interleukin-12 protein charge variant to be detected based on the chromatogram; the mobile phase adopted by the pH-IEC ion exchange chromatography comprises a mixed solution of citric acid and sodium phosphate; mobile phase a and mobile phase B were constituted by different mixing ratios. The pH-IEC ion exchange chromatography can be suitable for analyzing the charge variants of the recombinant human interleukin-12 protein with higher glycosylation and complex structure, solves the problems that the sample is denatured due to reagent or pretreatment, the separation of the charge isomers cannot be realized by the traditional high-salt elution-ion exchange chromatography and the like, can be used in the field of quality control of the medicines, ensures batch consistency of medicine production, and monitors the safety and effectiveness of clinical medication.

Description

Method for detecting recombinant human interleukin-12 protein charge variant and application

Technical Field

The invention belongs to the technical field of recombinant protein detection, and relates to a method for detecting a recombinant human interleukin-12 protein charge variant and application thereof.

Background

At present, most of protein drugs are produced by adopting a mammalian cell expression system, and aggregation, degradation and various post-translational modifications of products often occur in the processes of production, storage, transportation and the like of the products, so that variants of the products are generated. The heterogeneity caused by isoelectric point change and space charge distribution difference is charge heterogeneity, mainly comprising C-terminal lysine truncation, N-glutamine cyclization, N-glycosylation, oxidation, aggregation, aspartic acid deamination and isomerization, disulfide bond mismatch and the like. Based on variability calculations for known sites in a protein molecule, there may be as many as 2.58 million possible variants, thus yielding a range of potential product quality attributes.

In the guideline of ICH distribution, Q6B is divided into product-related substances and product-related impurities according to the influence degree of variants on the safety and effectiveness of products, and needs evaluation on the key quality attribute (CQA) of variant influence and safety. Isolation of these variations is crucial for the assessment of biophysical and biochemical properties, such as in vitro (potency, binding) and in vivo (toxicity, pharmacokinetics, immunogenicity) activity.

IEF separates the recombinant protein according to the pI characteristics of different charge variants of the recombinant protein, can only be qualitatively judged, and needs to denaturize and solubilize a sample. The iCIEF is used as a recombinant protein charge variant determination method, and is connected with a detector on the basis of IEF, so that quantitative analysis is realized, the detection time is short, and the defects of poor CE migration time and poor peak area reproducibility are overcome. But charge heterogeneity manifests itself as both a difference in charge and surface charge distributions. When the pI values of a plurality of variants are the same but the surface charge distribution of the protein is different, the IEC can separate the plurality of charge variants under non-denaturing conditions according to the difference of the charge distribution for quantitative analysis.

Publication No. CN106770597a discloses a CZE detection method for detecting erythropoietin charge heteroplasmon in a composition. The method adopts a capillary zone electrophoresis technology, and the scheme utilizes a capillary as a sample detection carrier, so that the reproducibility of migration time and peak area is poor due to the length of the capillary; the separation of CZE is not much helpful for the separation of charge heteroplasmons with similar molecular weights and cannot separate different charge heteroplasmons with charge distributions according to the charge-to-mass ratio of the protein; in addition, CZE requires the use of urea as a solubilizing reagent for the sample, which may result in sample denaturation.

Publication number CN109946410a discloses an ion exchange chromatography detection method for analyzing the charge heterogeneity of monoclonal antibodies. The method adopts an ion exchange chromatography technology, the scheme analyzes the protein to be detected through a high-salt elution-ion exchange chromatography, and based on the chromatogram, the content detection of the monoclonal antibody charge variant is completed, so that the effective separation of the target monoclonal antibody, the acid isomer, the alkaline isomer and other components is realized. However, the method is not suitable for the acidic recombinant proteins with high glycosylation and complex structure such as recombinant human interleukin-12, the charge heterogeneity of the recombinant human interleukin-12 is more complex, and because the isoelectric point range is wider and the isoelectric points of the components are closer, the elution time of salt ions to the components of the substance to be detected has no obvious difference, and the separation of the charge isomers can not be realized by the traditional high-salt elution-ion exchange chromatography.

The recombinant human interleukin-12 belongs to an acid glycoprotein, pI4.5-5.5, the range of isoelectric points is wide due to different glycosylation modes, a plurality of charge variants are abundant and have similar contents, and the IEC (International electrotechnical Commission) with the change of the concentration of salt ions cannot effectively separate the plurality of charge variants.

Disclosure of Invention

Based on the problems existing in the prior art, the invention aims to provide a method for detecting the recombinant human interleukin-12 protein charge variant; the invention also aims to provide the application of the method for detecting the recombinant human interleukin-12 protein charge variant in the production quality control of the recombinant human interleukin-12 protein.

The purpose of the invention is realized by the following technical means:

in one aspect, the invention provides a method for detecting a recombinant human interleukin-12 protein charge variant, which comprises the following steps:

analyzing the recombinant human interleukin-12 protein to be detected by a pH-IEC ion exchange chromatography to obtain a chromatogram, and determining the content of the recombinant human interleukin-12 protein charge variant to be detected based on the chromatogram;

the mobile phase adopted by the pH-IEC ion exchange chromatography comprises a mixed solution of citric acid and sodium phosphate; forming a mobile phase A and a mobile phase B by different mixing ratios;

wherein the pH value of the mobile phase A is 5.5-10.0, and is preferably 8.0; the pH value of the mobile phase B is 2.2-4.5, and is preferably 4.0.

The specific separation principle of the pH-IEC ion exchange chromatography is as follows: the pH gradient can change the degree of charge and deviation from isoelectric points of the detected substances, thereby affecting the binding capacity of molecules or ions with positive/negative charges and a chromatographic column stationary phase, and the difficulty degree during elution is changed, thereby changing the selectivity of separation and finally realizing the separation of each component.

The recombinant interleukin-12 protein is an acidic protein, the pI of the protein is 4.5-5.5, the charge variants are more, the difference of the pIs is smaller, 10 charge variants exist in the pI range of 4.5-5.5, the glycosylation modification degree is higher, the structure is complex, and the protein is sensitive to salt ion elution. The pH-IEC ion exchange chromatography solves the technical problem that the traditional high-salt elution-ion exchange chromatography and a salt concentration-mediated pH gradient method cannot realize the separation of acidic recombinant protein charge isomers with high glycosylation and complex structures, such as recombinant human interleukin-12, due to the fact that the pI difference among various complex charge variants is small and the acidic recombinant protein charge isomers are very sensitive to salt concentration to a certain extent, has the effect of quick, accurate and sensitive detection, and is particularly suitable for detecting the charge variants in the recombinant human interleukin-12 protein.

In the above method, preferably, the concentration of the citric acid is 5 to 50mmol/L, preferably 20mmol/L; the concentration of the sodium phosphate salt is 5-50 mmol/L, preferably 20mmol/L.

In the above method, preferably, the chromatography column used in the pH-IEC ion exchange chromatography comprises an anion exchange chromatography column.

In the above method, preferably, the chromatography column comprises a strong anion exchange chromatography column SAX.

In the above method, the elution time of the pH-IEC ion exchange chromatography is preferably 25 to 70min, more preferably 48min.

In the above method, preferably, the loading amount of the pH-IEC ion exchange chromatography is 10 to 50. Mu.l, the flow rate is 0.5 to 1.5ml/min, the column temperature is 20 to 60 ℃, and the detection wavelength is 280nm;

the elution procedure used in this method is as follows:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	60	40
48.00	0	100
			56.00	0	100
56.10	60	40
			70.00	60	40

. In the elution program, 0 minutes after the start of the run 60% a:40% B, 100% B48 minutes after the start of the run, the B phase increased uniformly over the 48 minutes. After 48min the column was equilibrated instead to bring the AB phase back to the initial level.

On the other hand, the invention also provides the application of the method for detecting the recombinant human interleukin-12 protein charge variant in the production quality control of the recombinant human interleukin-12 protein.

In the above application, preferably, when the detected spectrogram of the charge variant in the recombinant human interleukin-12 protein is consistent with the spectrogram of a physicochemical control product of the recombinant human interleukin-12 protein, the activity of the obtained recombinant human interleukin-12 protein biological product is not influenced or is influenced at a safe level, and the production can be released; when the detected spectrogram of the charge variant in the recombinant human interleukin-12 protein is inconsistent with the spectrogram of a physicochemical reference substance of the recombinant human interleukin-12 protein, the activity of the obtained recombinant human interleukin-12 protein biological product is influenced or the influence is in an unsafe level, the technological process for producing the biological product needs to be adjusted and improved, and the process can not be released until the charge variant of the produced biological product is in the safe level.

The above-mentioned "coincidence" means that the retention times of the liquid phase spectrograms are the same and the number of peaks is the same; "inconsistent" means that the liquid phase spectra have different retention times and different peak numbers.

On the other hand, the invention also provides application of the method for detecting the recombinant human interleukin-12 protein charge variant in detecting the recombinant human interleukin-12 protein charge variant.

The pH-IEC ion exchange chromatography can be suitable for analyzing the charge variants of acidic recombinant human interleukin-12 protein with higher glycosylation and complex structure, solves the problems that the sample is denatured due to reagent or pretreatment, the separation of charge isomers cannot be realized by the traditional high-salt elution-ion exchange chromatography and the like, can be used in the field of quality control of the recombinant human interleukin-12 protein, ensures batch consistency of drug production, and monitors the safety and effectiveness of clinical medication.

Drawings

FIG. 1 is a chromatogram obtained by pH-IEC ion exchange chromatography for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 1 of the present invention;

FIG. 2 is a chromatogram of Tris sodium salt-IEC ion exchange chromatography for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 2 of the present invention;

FIG. 3 is a chromatogram obtained by using MES sodium salt-IEC ion exchange chromatography to detect the charge heterogeneity of recombinant human interleukin-12 protein in example 2 of the present invention;

FIG. 4 is a chromatogram for cIEF method to detect the charge heterogeneity of recombinant human interleukin-12 protein in example 3 of the present invention;

FIG. 5 is a chromatogram obtained by pH-IEC ion exchange chromatography (wavelength 214 nm) for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 4 of the present invention;

FIG. 6 is a chromatogram obtained by pH-IEC ion exchange chromatography (WAX column) for detecting charge heterogeneity of recombinant human interleukin-12 protein in example 5;

FIG. 7 is a chromatogram obtained by pH-IEC ion exchange chromatography (with different pH ranges for mobile phase) for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 6 of the present invention;

FIG. 8 is a chromatogram for detecting the charge heterogeneity of the recombinant human interleukin-12 protein by pH-IEC ion exchange chromatography (linear gradient elution for 24 min) in example 7 under condition 1;

FIG. 9 is a chromatogram obtained by pH-IEC ion exchange chromatography (linear gradient elution for 48 min) for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 7 of the present invention under condition 2;

FIG. 10 is a chromatogram obtained by pH-IEC ion exchange chromatography (linear gradient elution for 72 min) for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 7 of the present invention;

FIG. 11 is an integrated chromatogram for detecting the charge heterogeneity of the recombinant human interleukin-12 protein by pH-IEC ion exchange chromatography (linear gradient elution for 48 min) in example 7 of the present invention, condition 2;

FIG. 12 is a chromatogram obtained by measuring the charge heterogeneity of the recombinant human interleukin-12 protein by pH-IEC ion exchange chromatography (linear step gradient elution for 48 min) in example 7 of the present invention under condition 2;

FIG. 13 is a chromatogram obtained by pH-IEC ion exchange chromatography (with different sample volumes) for detecting the charge heterogeneity of recombinant human interleukin-12 protein in example 8 of the present invention;

FIG. 14 is a chromatogram for detecting the charge heterogeneity of recombinant human interleukin-12 protein by ion exchange chromatography with the same pH-IEC (different batches) in example 9 of the present invention.

Detailed Description

The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.

Related name terminology interpretation:

ICH: the International coordination conference (International Council for Harmonization), according to the content of the coordination conference, chinese is usually translated into "International coordination conference for human drug registration technical requirement".

pI: isoelectric point (isoelectric point), pH at which one molecule or surface is not charged.

IEF: isoelectric focusing (isoelectrofocusing) is a method of electrophoresis in which a gradient of pH is created in a gel using a special buffer, and each protein migrates to a pH equal to its Isoelectric point, forming a narrow band.

CE: capillary electrophoresis (capillary electrophoresis) is a novel liquid phase separation technology which takes a capillary as a separation channel and takes a high-voltage direct-current electric field as a driving force.

And iCIEF: the Full-column imaging capillary isoelectric focusing electrophoresis (Full-column imaging capillary electrophoresis) is a kind of micro-separation technology which takes a capillary as a separation channel and a high-voltage direct-current electric field as a driving force, has no moving process after protein separation, and is a tool for representing protein charge heterogeneity.

C ZE: capillary zone electrophoresis (capillary electrophoresis) separates substances by the charge amount and size of ions.

ICE: ion Exchange Chromatography (Ion Exchange Chromatography), a type of high performance liquid Chromatography, is a liquid Chromatography method for analyzing anions and cations.

pH-IEC Ion Exchange Chromatography (pH Gradient Ion Exchange Chromatography), pH Gradient Ion Exchange Chromatography.

WAX: week Anion Exchange chromatography column (Week Anion Exchange).

SAX: strong Anion Exchange chromatography (Strong Anion Exchange).

In the following examples, reagents and instruments used are not specified by manufacturers, and are all conventional products commercially available. The main instruments, samples to be tested and experimental materials are shown in tables 1 to 3 below.

Table 1:

instrumentation apparatus	Model number	Device encoding
			PH meter	OHAUS 3100/F	GDZC-SY-YQ-0059
Suction filtration pump	Jinteng GM-0.33A	GDZC-SY-SB-0192
			Ultrasonic instrument	KQ-700DE	GDZC-SY-YQ-0040
High performance liquid chromatograph	Agilent1260	GDZC-SY-YQ-0003

Note: the main instrument and equipment information used are: ( The method comprises the following steps: the experimental instrument is qualified after being verified within the valid period )

Table 2:

table 3:

name(s)	Specification of	Batch number	Manufacturer of the product
				SAX-10 liquid chromatographic column	4×250mm	054997	Thermo
WAX-10 liquid chromatographic column	4×250mm	054999	Thermo
				Citric acid	500g	F20050005	Er Kang Zhiyao
Disodium hydrogen phosphate	500g	145746	fisher scientific

Example 1:

the implementation provides a method for detecting a recombinant human interleukin-12 protein charge variant by using a pH-IEC ion exchange chromatography, which comprises the following steps:

the chromatographic conditions are shown in table 4 below:

table 4:

conditional content	Name/index
		Liquid chromatograph	Agilent 1260
Detector/wavelength	DAD/280nm
		Chromatographic column	ProPac SAX-10(4*250mm)
Flow rate of flow	1ml/min
		Sample loading concentration	0.5mg/ml
Sample size	10μl
		Column temperature
	25℃

The collection time was 96min and the elution time was 60min. The elution procedure is shown in table 5 below:

table 5:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	100	0
08.00	100	0
			68.00	0	100
76.00	0	100
			76.10	100	0
96.00	100	0

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

And (3) mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

The resulting chromatogram is shown in FIG. 1. The result shows that the glycosylation structure of the recombinant human interleukin-12 protein sample is very much, and a plurality of peak signals can be detected by adopting the pH-IEC ion exchange chromatography.

Example 2:

the method for detecting the recombinant human interleukin-12 protein charge variant by adopting the high-salt-IEC ion exchange chromatography comprises the following specific steps:

the chromatographic conditions were the same as in example 1.

Condition 1:

the collection time was 96min, and the elution time was 60min. The elution procedure is shown in table 6 below:

table 6:

mobile phase A:10mM Tris-HCl, pH8.0;

and (3) mobile phase B:10mM Tris-HCl +1M NaCl, pH8.0.

The resulting chromatogram is shown in FIG. 2, and ion exchange chromatography using Tris sodium salt as the mobile phase failed to separate the charge variants.

Condition 2: the collection time was 96min and the elution time was 60min. The elution procedure is shown in table 7 below:

table 7:

Mobile phase A:20mM MES, pH8.0;

mobile phase B:20mM MES +1M NaCl, pH8.0.

The resulting chromatogram is shown in FIG. 3, and ion exchange chromatography using MES sodium salt as the mobile phase failed to separate the charge variants.

The results of the comparative example in FIG. 2 and FIG. 3 show that the pI of various complex charge variants of the recombinant human interleukin-12 protein sample have small difference, and the complex charge variants cannot be respectively eluted by the traditional salt concentration gradient separation method.

Example 3:

the method for detecting the recombinant human interleukin-12 protein charge variant by adopting the capillary electrophoresis method comprises the following specific steps:

the recombinant human interleukin-12 protein sample is measured by a full-column imaging capillary electrophoresis cIEF charge variant measuring method.

Sample preparation: 10 μ l sample + master mix using pI markers of 9.5, 7.0, 4.1, respectively.

Separating the capillary: the total length of the neutral coating capillary tube is 30cm, and the effective length of the neutral coating capillary tube is 20cm.

The obtained map is shown in figure 4, and the result shows that the recombinant protein sample has very many glycosylation structures, no obvious main peak and poor repeatability.

Example 4:

in the comparative example, the recombinant human interleukin-12 protein charge variant is detected and analyzed by changing the chromatographic measurement wavelength to be 214 nm. The method comprises the following specific steps:

the chromatographic conditions were the same as in example 1 except for the wavelength.

The collection time was 96min and the elution time was 60min. The elution procedure is shown in table 8 below:

table 8:

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

The resulting chromatogram is shown in FIG. 5. As a result, the chromatogram at the wavelength of 214nm revealed that the desired peak signal could not be detected, and it could be confirmed that the optimum signal value could be obtained at the wavelength of 280nm according to the present invention.

Example 5:

in the comparative example, the recombinant human interleukin-12 protein charge variant is detected and analyzed by replacing a chromatographic column with a weak anion exchange chromatographic column WAX-10. The method comprises the following specific steps:

the chromatographic conditions were the same as in example 1 except that the column was changed.

The collection time was 96min and the elution time was 60min. The elution procedure is shown in table 9 below:

table 9:

A mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

The resulting chromatogram is shown in FIG. 6. The result shows that when the weak anion exchange chromatographic column WAX-10 is used for analyzing the protein charge variant, the peak of each charge isomer variant cannot be well separated from the main peak, the expected effect cannot be achieved, and the quality control analysis is influenced, so that the method is not suitable for being adopted.

Example 6:

the comparative example detects and analyzes the recombinant human interleukin-12 protein charge variant by changing the pH value range of the mobile phase. The method comprises the following specific steps:

the chromatographic conditions were the same as in example 1.

Condition 1: the mobile phase B initial ratio was 0% (pH gradient 8.0 to 4.0).

The collection time was 78min and the elution time was 60min. The elution procedure is shown in table 10 below:

table 10:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	100	0
60.00	0	100
			68.00	0	100
68.10	100	0
			78.00	100	0

A mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

Condition 2: mobile phase B was initially at a ratio of 20% (pH gradient 7.2 to 4.0).

The collection time was 66min and the elution time was 48min. The elution procedure is shown in table 11 below:

table 11:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	80	20
48.00	0	100
			56.00	0	100
56.10	80	0
			66.00	80	0

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

Condition 3: mobile phase B was initially at a ratio of 40% (pH gradient 6.4 to 4.0).

The collection time was 54min and the elution time was 36min. The elution procedure is shown in table 12 below:

table 12:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	60	40
36.00	0	100
			44.00	0	100
44.10	60	40
			54.00	60	40

The resulting chromatogram is shown in FIG. 7. The result shows that the separation effect is kept unchanged, the peak emergence time is late when the initial proportion of the mobile phase B is 0 percent and 20 percent, and the detection time is long; the retention time is most suitable when the initial proportion of the mobile phase B is 40 percent, and the retention time is the shortest when the mobile phase B is expected to be used. The number of charge variants detected at an initial ratio of 40% for mobile phase B was identical to 0% indicating that each charge variant could be eluted from the column only if the pH gradient of the procedure covered the isoelectric point range of the recombinant protein (pI of this sample was 4.5-5.5). Therefore, the detection method can separate the protein sample with the pI between 3 and 9 and is not limited by the pH value of the protein.

Example 7:

the comparative example detects and analyzes the recombinant human interleukin-12 protein charge variant by changing the elution time. The method comprises the following specific steps:

the chromatographic conditions were the same as in example 1.

Condition 1:

the collection time was 46min and the elution time was 24min. The elution procedure is shown in table 13 below:

table 13:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	60	40
24.00	0	100
			32.00	0	100
32.10	60	40
			46.00	60	40

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

Condition 2:

the collection time was 70min and the elution time was 48min. The elution procedure is shown in table 14 below: table 14:

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

Condition 3:

the collection time was 94min and the elution time 72min. The elution procedure is shown in table 15 below: table 15:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	60	40
72.00	0	100
			80.00	0	100
80.10	60	40
			94.00	60	40

Condition 4:

the collection time was 54min and the elution time was 40min. The elution procedure is shown in table 16 below: table 16:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			00.00	60	40
19.40	27.6	72.4
			21.40	27.6	72.4
25.50	24.5	75.5
			27.50	24.5	75.5
40.00	0.0	100.0
			40.10	60	40
54.00	60	40

Mobile phase A:20mM citric acid +20mM disodium hydrogen phosphate, pH8.0

Mobile phase B:20mM citric acid +20mM disodium hydrogen phosphate, pH4.0

The chromatograms obtained are shown in FIGS. 8 to 12. The results show that the degree of separation is lower at 24min elution time (fig. 8); the signal peak detected at 72min of elution time was weaker (FIG. 10); the separation degree is highest when the elution time is 48min, the number of detected peaks is the largest, the ideal separation effect is obtained (figure 9), 10 variants of the target protein can be separated under the condition, the content is similar, and no obvious main peak is generated (figure 11). The inventors have tried to extend the elution time at the peak to obtain better separation, and the results showed that the stepwise gradient elution failed to obtain better separation and the number of separable variants decreased (fig. 12).

Therefore, the effect of the method is greatly influenced by elution time and elution gradient, and a step gradient elution mode cannot achieve better effect. The elution time can be linear gradient elution for 25-70 min, and the signal peak is weak when the time exceeds 70min, so that all charge variants cannot be detected. The optimal elution time is 48min.

Example 8:

in this comparative example, the charge variants of recombinant human interleukin-12 protein were examined and analyzed by changing the sample volumes (10. Mu.l, 20. Mu.l, 40. Mu.l). The method comprises the following specific steps:

the chromatographic conditions were the same as in example 1 except for the amount of the sample.

The elution procedure was the same as in example 7, condition 2.

The resulting chromatogram is shown in FIG. 13. The result shows that the separation degree of different sample volumes is unchanged, the reproducibility of retention time is better, and the method is stable and reliable and can be realized. Limited by the performance of the chromatographic column, avoids false peaks caused by excessive concentration coagulation, and the sample injection amount is not more than 50 mu l.

Example 9: inter-batch consistency testing

The detection method is the same as example 7, condition 2, and charge variants of 3 batches of recombinant human interleukin-12 stock solution in the same process as CHH201804002, CHH201804003 and CHH201805004 are analyzed, and the obtained chromatogram is shown in FIG. 14. PBS is used for adjusting 3 batches of stock solution to the same concentration, the sample amount is 10 mug, the batch consistency is good, the percentage content of each component of the monoclonal antibody can be detected (Table 17), and the ideal analysis effect is obtained.

Table 17:

as can be seen from the experimental data in table 17: the consistency among batches is good, and an ideal analysis effect is obtained.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. The application of the method for detecting the recombinant human interleukin-12 protein charge variant in the production quality control of the recombinant human interleukin-12 protein charge variant is disclosed, wherein the method for detecting the recombinant human interleukin-12 protein charge variant comprises the following steps:

analyzing the protein to be detected by a pH-IEC ion exchange chromatography to obtain a chromatogram, and determining the content of the recombinant human interleukin-12 protein charge variant to be detected based on the chromatogram;

wherein the pH value of the mobile phase A is 5.5-10.0; the pH value of the mobile phase B is 2.2-4.5;

the elution procedure used in this method is as follows:

time, min Mobile phase A% Mobile phase B,% 00.00 60 40 48.00 0 100 56.00 0 100 56.10 60 40 70.00 60 40

The chromatographic column adopted by the pH-IEC ion exchange chromatography comprises a strong anion exchange chromatographic column SAX;

the sample loading amount of the pH-IEC ion exchange chromatography is 10-50 mu l, the flow rate is 0.5-1.5ml/min, the column temperature is 20-60 ℃, and the detection wavelength is 280nm.

2. Use according to claim 1, wherein the mobile phase a has a pH value of 8.0.

3. Use according to claim 1, wherein the mobile phase B has a pH value of 4.0.

4. The use according to claim 1, wherein the concentration of citric acid is 5-50 mmol/L.

5. The use according to claim 4, wherein the citric acid concentration is 20mmol/L.

6. The use according to claim 1, wherein the concentration of the sodium phosphate salt is 5 to 50mmol/L.

7. Use according to claim 6, wherein the concentration of the sodium phosphate salt is 20mmol/L.

8. The use of claim 1, wherein when the detected spectrum of the charge variant of the recombinant human interleukin-12 protein charge variant is consistent with the spectrum of a physicochemical control substance of the recombinant human interleukin-12 protein charge variant, the activity of the biological product of the recombinant human interleukin-12 protein charge variant is not affected or affected at a safe level, and the production can be released; when the detected spectrogram of the charge variant in the recombinant human interleukin-12 protein charge variant is inconsistent with the spectrogram of a physicochemical reference substance of the recombinant human interleukin-12 protein charge variant, the activity of the obtained recombinant human interleukin-12 protein charge variant biological product is influenced or influenced to be in an unsafe level, the technological process for producing the biological product needs to be adjusted and improved, and the discharge can not be released until the charge variant of the produced biological product is in the safe level.