CN114636781B - Method for evaluating sialylation level of glycoprotein oligosaccharide - Google Patents
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Abstract
The invention discloses a method for evaluating the sialylation level of glycoprotein oligosaccharide. The method comprises the following steps: glycoprotein denaturation, collection and concentration, enzyme digestion of sugar chains, precipitation, sugar chain enrichment and ion chromatography. The invention utilizes the high-efficiency anion exchange chromatography to detect the sialylation level of glycoprotein oligosaccharide, overcomes the problems of complex operation steps, low high sialylation sugar chain marking efficiency and the like of the traditional marking, can accurately analyze the oligosaccharide map of glycoprotein, is more beneficial to accurately analyze each sialylation component, and is suitable for evaluating the sialylation level of glycoprotein oligosaccharide.
Description
Technical Field
The invention relates to the field of protein medicine bioengineering and technology, and relates to a method for evaluating sialylation level of glycoprotein oligosaccharide. In particular to an evaluation method of the sialylation level of recombinant human vascular endothelial cell growth factor receptor Fc (VEGFR-Fc) fusion protein oligosaccharide.
Background
Glycosylation is one of the most common post-translational modifications of proteins and can be classified into N-glycosylation and O-glycosylation depending on the manner of attachment, where N-glycosylation is the-NH-on the sugar chain and Asn residues in the amino acid sequence of the encoded protein Asn-X-Ser/Thr (X is an amino acid other than Pro) 2 In connection, O-glycosylation is the attachment of the sugar chain to the-OH group on the amino acid sequence Ser/Thr residue of the encoded protein. At present, therapeutic protein drugs are mostly expressed by gene recombinant cells, and the glycosylation type and the glycosylation pattern thereofThe level has different effects on the efficacy, half-life, stability, safety and the like of the medicine in vivo. For example, for monoclonal antibodies or fusion proteins, lower levels of sialic acid at the ends affect the pharmacokinetics (pK), leading to rapid clearance in vivo and reduced half-life, and therefore sialic acid levels are an important indicator for assessing glycosylation levels. Wherein the oligosaccharide profile can display the glycoform distribution of the glycoprotein with different sialic acid numbers, and calculating the glycoform proportion of the different sialic acid numbers, and the Z value is an important parameter for representing the integral sialylation degree of the glycoprotein. The detection program is mainly used for representing the sialylation degree of the recombinant therapeutic glycoprotein and examining the glycosylation stability among batches.
Currently, methods for determining oligosaccharide profile (Z value) mainly include high performance liquid chromatography and ion chromatography. The method for measuring the Z value by the high performance liquid chromatography needs to carry out 2-AA/2-AB labeling, has the problems of complex operation steps (2-AB method), low labeling efficiency of high sialylated sugar chains (2-AA method) and the like, and the ion chromatography detection principle adopted by the method is based on different charges of sugar chains containing different amounts of sialic acid, the oligosaccharides containing different sialic acid numbers are separated by using an anion chromatographic column, and then the oligosaccharides are detected by using a high-sensitivity integral pulse ampere detector, so that a sample does not need to be labeled, the operation is simple, the sensitivity is high, the sialylation degree of VEGFR-Fc fusion protein can be reflected more truly, the final detection result is embodied by the Z value, and the Z value is defined as the average number of sialic acid in each oligosaccharide. The method is favorable for deep analysis of sialylation degree of complex glycoprotein, is favorable for researching the difference of sialylation degree between different charge isomers, and is favorable for quality control of different sialylated glycoprotein in the production process.
Disclosure of Invention
The recombinant human VEGFR-Fc fusion protein is a homodimeric glycoprotein, is expressed by Chinese hamster ovary cells (CHO cells), and is obtained through high purification and virus inactivation steps. The protein contains multiple glycosylation modification sites, has multiple charge isomers, and is shown as not less than 12 bands on IEF (see figure 1), and the difference between different bands is mainly caused by sialic acid. To ensure uniformity of sialylation degree for each production lot, a stable production process and an effective sialylation analysis method are required. In order to further carry out quality study on the product, finely control the purification process and improve the product quality, the glycoprotein is denatured and reduced by guanidine hydrochloride and DTT, enzyme digestion, alcohol precipitation, concentration drying and resuspension are carried out on the denatured protein by using glycosidase F, and oligosaccharide map detection analysis is carried out on the enriched sugar chains by combining a high-efficiency anion exchange chromatography-integral pulse amperometric detector (HPAEC-PAD) to guide the collection of the target protein in the purification process, so that the aim of obtaining the high-quality glycoprotein medicine is fulfilled.
In view of the above, the invention aims to provide a method for evaluating the sialylation level of glycoprotein oligosaccharide, which overcomes the problems of complex operation steps of a 2-AB method, high sialylation sugar chain marking efficiency of a 2-AA method, and the like, can analyze glycoprotein oligosaccharide maps (Z values), can accurately analyze the sialylation degree of glycoprotein and the difference of different sialylation oligosaccharides, is beneficial to the collection of target protein in the purification process, and achieves the aim of obtaining high-quality glycoprotein drugs.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a method for evaluating the sialylation level of glycoprotein oligosaccharide, which adopts high-performance ion chromatography for analysis and specifically comprises the following steps:
(1) Carrying out denaturation and reduction on the protein by using a denaturant and a reducing agent, and carrying out alkylation treatment on the denatured and reduced protein;
(2) Changing the liquid of the alkylated protein, and collecting the protein into a centrifuge tube for concentration;
(3) Enzyme cutting is carried out on the concentrated protein by using glycosidase F;
(4) Adding an organic solvent into the protein solution after enzyme digestion, precipitating, centrifuging and taking supernatant;
(5) Concentrating and drying the supernatant obtained in the step (4), and re-suspending to obtain an oligosaccharide solution to be detected;
(6) The oligosaccharide solution in step (5) was subjected to detection analysis using a high performance anion exchange chromatography-integrated pulse amperometric detector.
Wherein the glycoprotein is a glycoprotein having multiple glycosylation sites.
Wherein the glycoprotein has a plurality of charge isomers.
Wherein the glycoprotein is recombinant human VEGFR-Fc fusion protein.
Wherein the denaturing agent used in step 1 is guanidine hydrochloride or urea, of which guanidine hydrochloride is preferred.
Wherein the buffer system in the step 1 is Tris-HCl buffer solution, the reducing agent is dithiothreitol, and the alkylating agent is iodoacetamide.
In one embodiment, denaturation can be achieved by: and (3) taking a proper amount of recombinant human VEGFR-Fc fusion protein, adding 1M Tris-HCl buffer solution, 8M guanidine hydrochloride and 1M DTT to ensure that the protein concentration in a final system is 0.5-1mg/ml, the final Tris-HCl concentration is 50mM, the final guanidine hydrochloride concentration is 6M and the final DTT concentration is 10mM, uniformly mixing, and then carrying out water bath at 56 ℃ for 30min.
Wherein the step 1 uses Dithiothreitol (DTT) solution to reduce glycoprotein; adding a DTT solution with the concentration of 0.5M-1M, and carrying out reduction treatment on the protein, wherein the final concentration of the DTT is 2-25mM; further preferably the final concentration of DTT is 5-20mM; in some embodiments the final concentration of DTT is 2mM, 10mM or 25mM.
Wherein step 1 is an alkylation treatment using Iodoacetamide (IAM), wherein IAM is used at a concentration of 5-62.5 mM, in some embodiments at 5mM, 25mM, 62.5mM.
Wherein in step 2, a desalting column or an ultrafiltration centrifuge tube is used for liquid exchange, preferably a desalting column, more preferably a Hitrap desalting column.
Wherein the final concentration of glycosidase F used in step 3 is 2-20U/. Mu.l; it is further preferred that the final concentration of glycosidase F is 4-10U/. Mu.l; more preferably, the final concentration of glycosidase F is 5U/. Mu.l.
Among the organic solvents used for protein precipitation in step 4 are methanol, ethanol or acetone, preferably ethanol or acetone, and in some embodiments ethanol is further preferred.
Wherein the volume ratio of the organic solvent used is 50% to 80%, more preferably 65% to 75%, and in certain embodiments 50%, 75% or 80%.
In some embodiments the volume ratio of ethanol used is 75%.
In one embodiment, ion chromatography uses a Thermo Scientific ICS-5000+ DC system, chromatography columns using Dionex CarboPac PA (3 x 250 mm), and Guard columns using Dionex CarboPac PA Guard (3 x 50 mm). And then collecting the sample and analyzing and processing the data.
The ion chromatography adopts gradient elution, and the elution conditions are as follows:
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
elution settings:
a method of evaluating the sialylation level of a glycoprotein oligosaccharide comprising the steps of: (1) Carrying out denaturation and reduction treatment on the protein through guanidine hydrochloride and Dithiothreitol (DTT), and carrying out alkylation treatment on the denatured and reduced protein; (2) Changing liquid of the alkylated protein and the desalting column, collecting the liquid into a centrifuge tube, and concentrating the liquid to a certain volume; (3) Carrying out enzyme digestion on the desalted and concentrated protein by using glycosidase F; (4) Adding 75% ethanol by volume into the collected and concentrated protein, precipitating, centrifuging, and collecting supernatant to obtain oligosaccharide solution without protein; (5) Concentrating and drying the oligosaccharide solution, and re-suspending to a certain volume to obtain an enriched oligosaccharide solution; (6) Oligosaccharide profile detection analysis was performed on the enriched sugar chains using a high performance anion exchange chromatography-integrated pulse amperometric detector (HPAEC-PAD) and the Z value was calculated according to the formula:
Z=（A1×1+A2×2+A3×3+A4×4）/（A0+A1+A2+A3+A4）；
wherein:
a0 is the percentage of the total peak area occupied by the 0-sialylation peak area;
a1 is the percentage of 1-sialylation peak area to total peak area;
a2 is the percentage of the 2-sialylation peak area to the total peak area;
a3 is the percentage of the 3-sialylation peak area to the total peak area;
a4 is the percentage of the 4-sialylation peak area to the total peak area.
The method for detecting the high-efficiency anion exchange chromatography is further as follows:
the dried oligosaccharide was added with 200. Mu.l of ultrapure water, and after complete dissolution, centrifuged at 13000rpm for 10min, and the supernatant was taken for ion chromatography. Ion chromatograph was Thermo Scientific ICS-5000+dc system, using Dionex CarboPac PA200 (3 x 250 mm) column, and after sample collection, the different sialylated peaks were integrated and data analyzed.
The ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min;
column temperature: 30 ℃;
sample injection volume: 25 μl full loop sample injection;
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
table 1: ion chromatography elution gradient
The method directly detects and analyzes the enriched oligosaccharides by adopting the high-efficiency anion exchange chromatographic column with high resolution to separate the oligosaccharide map from the enriched sugar chains and using the integral pulse ampere detector to detect the separated oligosaccharides, thereby reducing the influence of the low labeling efficiency on the result of the oligosaccharides and being beneficial to truly reflecting the oligosaccharide map (Z value) of glycoprotein.
According to the invention, through adopting guanidine hydrochloride denaturation, a desalting column for liquid exchange, enzyme digestion, precipitation and sugar chain enrichment of glycosidase F, complicated steps of labeling sugar chains by 2-AB/2-AA, purifying sugar chains and the like are avoided, expensive consumables and equipment are not needed, samples do not need to be labeled, and the method is convenient to operate, and can simply and rapidly finish enzyme digestion and collect sugar chains.
The invention separates the oligosaccharide by protein denaturation, sugar chain enzyme digestion and enrichment and combining with a high-efficiency anion exchange chromatographic column, and then uses an integral pulse ampere detector to detect the separated oligosaccharide. Compared with the prior art, the method has the advantages that firstly, the oligosaccharide treatment process does not need to be marked, the operation is simple, the sialylation degree of glycoprotein can be reflected more truly, secondly, the method adopts the high-efficiency anion exchange chromatography-integral pulse amperometric detector to carry out oligosaccharide map detection analysis on the enriched sugar chains, has high sensitivity, can reflect the sialylation degree of complex glycoprotein more truly, is beneficial to researching the glycoform difference among different sialylation isomers, is beneficial to carrying out quality control on different sialylation isomers in the production process, and has good referential significance on glycoprotein oligosaccharide map analysis with multiple sialylation isomers.
Drawings
Fig. 1: recombinant human VEGFR-Fc fusion protein IEF electropherograms;
fig. 2: recombinant human VEGFR-Fc fusion protein oligosaccharide profile (ion chromatography);
fig. 3: recombinant human VEGFR-Fc fusion protein oligosaccharide profile (high performance liquid chromatography, 2-AB method);
fig. 4: recombinant human VEGFR-Fc fusion protein oligosaccharide profile (high performance liquid chromatography, 2-AA method);
fig. 5: recombinant human VEGFR-Fc fusion protein oligosaccharide profile (ion chromatography, urea denaturation);
fig. 6: recombinant human VEGFR-Fc fusion protein oligosaccharide profile (ion chromatography, acetone precipitation);
fig. 7: oligosaccharide profile of recombinant human VEGFR-Fc fusion protein (ion chromatography, ultrafiltration exchange).
Detailed Description
In order to further illustrate the present invention, a technical method in the embodiments of the present invention will be clearly and completely described below in connection with the specific embodiments of the present invention. Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1 analysis of oligosaccharide maps (Z values) of recombinant human VEGFR-Fc fusion proteins
(1) Denaturation and reduction
Taking 1.5ml of EP tube, respectively adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mug protein and a proper amount of ultrapure water, so that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of guanidine hydrochloride is 6M, and the final concentration of DTT is 10mM, the total reaction system is 300 mul, setting blank samples at the same time, sequentially adding the reagents, uniformly mixing, and carrying out water bath at 56 ℃ for 30min;
(2) Alkylation
Cooling the sample to room temperature, respectively adding 7.5 mu l of 1M IAM, uniformly mixing, and carrying out light-shielding reaction at room temperature for 45min;
(3) Desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280nm. Firstly, taking 275 μl of 6M guanidine hydrochloride, pre-washing a sample injection needle and a pipeline, then sequentially injecting 275 μl according to the sample sequence, collecting protein peaks, and collecting about 600 μl;
(4) Concentrating
Concentrating the collected sample to 300 mu l, if the concentration is less than 300 mu l, and using ultrapure water to fix the volume to 300 mu l;
(5) Sugar cutter
Collecting the concentrated sample, adding glycosidase F3 μl, mixing, and performing water bath enzyme digestion at 37deg.C for 15-18 hr;
(6) Sugar chain enrichment
Adding 0.9ml of ice ethanol stored at-30deg.C into the sample, mixing, precipitating with ethanol at-30deg.C for 15min, centrifuging at 13000rpm for 15min at 4deg.C with a refrigerated centrifuge;
(7) Sugar chain drying
Taking the supernatant after centrifugation, and putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) Ion chromatography detection
Adding 200 μl of ultrapure water into the dried oligosaccharide, completely dissolving, centrifuging at 13000rpm for 10min, collecting supernatant, performing ion chromatography detection, using ion chromatography with Thermo Scientific ICS-5000 +DC system, using Dionex CarboPac PA200 (3×250mm) column, integrating different sialylated peaks after sample collection, and performing data analysis;
the ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min;
column temperature: 30 ℃;
sample injection volume: 25 μl full loop sample injection;
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
table 1: ion chromatography elution gradient
(9) Experimental results
Determining the outlet position of a solvent peak according to a blank sample, deleting the solvent peak and the gradient peak, and combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, and the fifth main peak cluster is a 4-sialylation peak, and the graph is shown in figure 2.
The Z value is calculated according to the formula z= (a1×1+a2×2+a3×3+a4×4)/(a0+a1+a2+a3+a4), wherein: a0 is the percentage of the total peak area occupied by the 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of the 2-sialylation peak area to the total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the percentage of 4-sialylation peak area to total peak area, and the results are shown in table 2:
TABLE 2 results of Z values for recombinant human VEGFR-Fc fusion proteins
In addition, the method for measuring oligosaccharide profile (Z value) mainly includes high performance liquid chromatography and ion chromatography. The method for measuring Z value by high performance liquid chromatography requires 2-AA/2-AB labeling, has complex operation steps (after sugar chain drying, labeling, purification, re-enrichment and re-drying are required), has low resolution (2-AB method), and as shown in figure 3, each sialylated peak shows 1-2 peaks respectively, the resolution is not high, and each sialylated peak detected by ion chromatography basically shows 4-10 peaks respectively, and the resolution is high; the high sialylated sugar chain labelling efficiency was low (2-AA method), as shown in FIG. 4, and the Z-value results are shown in Table 3. The 2-AA method labelling of high sialyloligosaccharides, such as 3SA% oligosaccharide results (2.4%) were significantly lower than 5.7% by ion chromatography, and 2SA% oligosaccharide results (22.6%) were also lower than 32.1% by ion chromatography, ultimately resulting in a lower Z-value (2-AA method Z-value of 0.95, significantly lower than ion chromatography Z-value of 1.23). Thus, the advantage of detecting oligosaccharide maps by ion chromatography is more revealed.
TABLE 3 results of Z values for recombinant human VEGFR-Fc fusion protein (2-AB/2-AA method)
Example 2 analysis of oligosaccharide maps (Z values) of recombinant human VEGFR-Fc fusion proteins (Urea denaturation)
(1) Denaturation and reduction
Taking 1.5ml of EP tube, respectively adding 1M Tris-HCl, 10M urea, 1M DTT, 200 mug protein and proper amount of ultrapure water, so that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of urea is 8M, and the final concentration of DTT is 10mM, the total reaction system is 300 mul, setting blank samples at the same time, sequentially adding the reagents, uniformly mixing, and carrying out water bath at 56 ℃ for 30min;
(2) Alkylation
Cooling the sample to room temperature, respectively adding 7.5 mu l of 1M IAM, uniformly mixing, and carrying out light-shielding reaction at room temperature for 45min;
(3) Desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280nm. 275 μl of 8M urea is sampled, and the sample injection needle and the pipeline are pre-washed. Then 275 μl of sample is injected according to the sample sequence, protein peaks are collected, and the collection volume is about 600 μl;
(4) Concentrating
Concentrating the collected sample to 300 mu l, if the concentration is less than 300 mu l, and using ultrapure water to fix the volume to 300 mu l;
(5) Sugar cutter
Collecting the concentrated sample, adding glycosidase F3 μl, mixing, and performing water bath enzyme digestion at 37deg.C for 15-18 hr;
(6) Sugar chain enrichment
Adding 0.9ml of ice ethanol stored at-30deg.C into the sample, mixing, precipitating with ethanol at-30deg.C for 15min, centrifuging at 13000rpm for 15min at 4deg.C with a refrigerated centrifuge;
(7) Sugar chain drying
Taking the supernatant after centrifugation, and putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) Ion chromatography detection
The dried oligosaccharide was added with 200. Mu.l of ultrapure water, and after complete dissolution, centrifuged at 13000rpm for 10min, and the supernatant was taken for ion chromatography. The ion chromatograph is a Thermo Scientific ICS-5000+DC system, a chromatographic column uses Dionex CarboPac PA200 (3 x 250 mm), and after sample collection, different sialylated peaks are integrated and data analysis is performed;
the ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min;
column temperature: 30 ℃;
sample injection volume: 25 μl full loop sample injection;
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
table 4: ion chromatography elution gradient
(9) Experimental results
Determining the outlet position of a solvent peak according to a blank sample, deleting the solvent peak and the gradient peak, and combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, and the fifth main peak cluster is a 4-sialylation peak, and the graph is shown in figure 5.
The Z value is calculated according to the formula z= (a1×1+a2×2+a3×3+a4×4)/(a0+a1+a2+a3+a4), wherein: a0 is the percentage of the total peak area occupied by the 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of the 2-sialylation peak area to the total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the percentage of 4-sialylation peak area to total peak area, and the results are shown in table 5:
TABLE 5 recombinant human VEGFR-Fc fusion protein Z value results (Urea denaturation)
Example 3 analysis of oligosaccharide profile (Z-value) of recombinant human VEGFR-Fc fusion protein (acetone precipitation)
(1) Denaturation and reduction
Taking 1.5ml of EP tube, respectively adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mug protein and a proper amount of ultrapure water, so that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of guanidine hydrochloride is 6M, and the final concentration of DTT is 5mM, the total reaction system is 300 mul, setting blank samples at the same time, sequentially adding the reagents, uniformly mixing, and carrying out water bath at 56 ℃ for 30min;
(2) Alkylation
Cooling the sample to room temperature, respectively adding 3.75 mu l of 1M IAM, uniformly mixing, and carrying out light-shielding reaction at room temperature for 45min;
(3) Desalting column liquid exchange
Connecting the desalting column with a high performance liquid chromatograph, balancing the chromatographic column with desalting buffer solution for 30min, and preparing sample injection under the chromatographic conditions of flow rate: 0.8ml/min, detection wavelength: 280nm. 275 μl of 6M guanidine hydrochloride was sampled, and the needle and tubing were pre-washed. Then 275 μl of sample is injected according to the sample sequence, protein peaks are collected, and the collection volume is about 600 μl;
(4) Concentrating
Concentrating the collected sample to 300 mu l, if the concentration is less than 300 mu l, and using ultrapure water to fix the volume to 300 mu l;
(5) Sugar cutter
Collecting the concentrated sample, adding glycosidase F6 μl, mixing, and performing water bath enzyme digestion at 37deg.C for 15-18 hr;
(6) Sugar chain enrichment
Adding 1.2ml of acetone stored at-30deg.C into the above sample, mixing, precipitating at-30deg.C for 15min, centrifuging at 13000rpm for 15min at 4deg.C with a refrigerated centrifuge;
(7) Sugar chain drying
Taking the supernatant after centrifugation, and putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(8) Ion chromatography detection
The dried oligosaccharide was added with 200. Mu.l of ultrapure water, and after complete dissolution, centrifuged at 13000rpm for 10min, and the supernatant was taken for ion chromatography. The ion chromatograph is a Thermo Scientific ICS-5000+DC system, a chromatographic column uses Dionex CarboPac PA200 (3 x 250 mm), and after sample collection, different sialylated peaks are integrated and data analysis is performed;
the ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min;
column temperature: 30 ℃;
sample injection volume: 25 μl full loop sample injection;
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
table 6: ion chromatography elution gradient
(9) Experimental results
Determining the peak position of a solvent peak according to a blank sample, deleting the solvent peak and the gradient peak, and combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, and the fifth main peak cluster is a 4-sialylation peak, and the graph is shown in figure 6.
The Z value is calculated according to the formula z= (a1×1+a2×2+a3×3+a4×4)/(a0+a1+a2+a3+a4), wherein: a0 is the percentage of the total peak area occupied by the 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of the 2-sialylation peak area to the total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the percentage of 4-sialylation peak area to total peak area, and the results are shown in table 7:
TABLE 7 recombinant human VEGFR-Fc fusion protein Z value results (acetone precipitation)
Example 4 analysis of oligosaccharide maps (Z values) of recombinant human VEGFR-Fc fusion proteins (ultrafiltration exchange solutions)
(1) Denaturation and reduction
Taking 1.5ml of EP tube, respectively adding 1M Tris-HCl, 8M guanidine hydrochloride, 1M DTT, 200 mug protein and a proper amount of ultrapure water, so that the final concentration of Tris-HCl in a final system is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of DTT is 25mM, the total reaction system is 300 mul, setting blank samples at the same time, sequentially adding the reagents, uniformly mixing, and carrying out water bath at 56 ℃ for 30min;
(2) Alkylation
Cooling the sample to room temperature, adding 18.75 mu l of 1M IAM respectively, mixing uniformly, and reacting for 45min at room temperature in a dark place;
(3) Ultrafiltration liquid exchange
Ultrafiltering the sample with 0.5ml 10kD ultrafilter tube for 4 times to obtain concentrated solution of about 50 μl, and fixing the volume of the ultrafilter buffer solution to 300 μl;
(4) Sugar cutter
Taking a sample subjected to ultrafiltration liquid exchange, adding 3 μl of glycosidase F, mixing, and performing water bath enzyme digestion at 37 ℃ for 15-18h;
(5) Sugar chain enrichment
Adding 0.9ml of ethanol stored at-30deg.C into the above sample, mixing, precipitating with ethanol at-30deg.C for 15min, centrifuging at 13000rpm for 15min at 4deg.C with a refrigerated centrifuge;
(6) Sugar chain drying
Taking the supernatant after centrifugation, and putting the supernatant into a freeze concentration centrifuge for concentration until the supernatant is completely dried;
(7) Ion chromatography detection
The dried oligosaccharide was added with 200. Mu.l of ultrapure water, and after complete dissolution, centrifuged at 13000rpm for 10min, and the supernatant was taken for ion chromatography. The ion chromatograph is a Thermo Scientific ICS-5000+DC system, a chromatographic column uses Dionex CarboPac PA200 (3 x 250 mm), and after sample collection, different sialylated peaks are integrated and data analysis is performed;
the ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min;
column temperature: 30 ℃;
sample injection volume: 25 μl full loop sample injection;
mobile phase a:0.02M sodium hydroxide solution;
mobile phase B:0.1M sodium hydroxide solution;
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution;
table 6: ion chromatography elution gradient
(8) Experimental results
Determining the outlet position of a solvent peak according to a blank sample, deleting the solvent peak and the gradient peak, and combining component peaks in each peak cluster, wherein the first main peak cluster is a 0-sialylation peak, the second main peak cluster is a 1-sialylation peak, the third main peak cluster is a 2-sialylation peak, the fourth main peak cluster is a 3-sialylation peak, and the fifth main peak cluster is a 4-sialylation peak, and the graph is shown in figure 7.
The Z value is calculated according to the formula z= (a1×1+a2×2+a3×3+a4×4)/(a0+a1+a2+a3+a4), wherein: a0 is the percentage of the total peak area occupied by the 0-sialylation peak area; a1 is the percentage of 1-sialylation peak area to total peak area; a2 is the percentage of the 2-sialylation peak area to the total peak area; a3 is the percentage of the 3-sialylation peak area to the total peak area; a4 is the percentage of 4-sialylation peak area to total peak area, and the results are shown in table 8:
TABLE 8 recombinant human VEGFR-Fc fusion protein Z value results (Ultrafiltration fluid exchange)
Claims (15)
1. A method for evaluating the sialylation level of a glycoprotein oligosaccharide, which method uses high performance ion exchange chromatography for analysis, comprising the steps of:
(1) Carrying out denaturation and reduction on the protein by using a denaturant and a reducing agent, and carrying out alkylation treatment on the denatured and reduced protein;
(2) Changing the liquid of the alkylated protein, and collecting the protein into a centrifuge tube for concentration;
(3) Enzyme cutting is carried out on the concentrated protein by using glycosidase F;
(4) Adding an organic solvent into the protein solution after enzyme digestion, precipitating, centrifuging and taking supernatant;
(5) Concentrating and drying the supernatant obtained in the step (4), and re-suspending to obtain an oligosaccharide solution to be detected;
(6) Performing detection analysis on the oligosaccharide solution in step (5) using a high performance anion exchange chromatography-integrated pulse amperometric detector, wherein the chromatographic column is Dionex CarboPac PA 200.3×250mm and the Guard column is Dionex CarboPac PA 200.200 Guard 3×50 mm;
wherein the high-efficiency ion exchange chromatography adopts gradient elution, and the elution conditions are as follows:
mobile phase a: a solution of 0.02M sodium hydroxide,
mobile phase B: a solution of 0.1M sodium hydroxide,
mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution,
the elution gradient was as follows:
Wherein the glycoprotein is recombinant human VEGFR-Fc fusion protein.
2. The method according to claim 1, wherein the denaturing agent used in step (1) is guanidine hydrochloride or urea.
3. The method according to claim 1, wherein the buffer system in step (1) is Tris-HCl buffer, the reducing agent is dithiothreitol, and the alkylating agent is iodoacetamide.
4. The method of claim 1, wherein the denaturation of step (1) is achieved by: taking a proper amount of recombinant human VEGFR-Fc fusion protein, adding 1M Tris-HCl buffer solution, 8M guanidine hydrochloride and 1M dithiothreitol to ensure that the concentration of the protein in a final system is 0.5-1mg/ml, the final concentration of Tris-HCl is 50mM, the final concentration of guanidine hydrochloride is 6M and the final concentration of dithiothreitol is 10mM, uniformly mixing, and then carrying out water bath at 56 ℃ for 30min;
in the step (1), dithiothreitol solution is used for carrying out reduction treatment on glycoprotein, the concentration of the dithiothreitol solution is added to be 0.5M-1M, and the final concentration of the dithiothreitol for carrying out reduction treatment on the protein is 2-25mM; the step (1) is performed by alkylation with iodoacetamide, wherein the concentration of iodoacetamide used is 5-62.5. 62.5mM.
5. The method according to claim 4, wherein the final concentration of dithiothreitol for the reduction treatment of the protein in step (1) is 5 to 20mM.
6. The method of claim 1, wherein the liquid exchange is performed in step (2) using a desalting column or an ultrafiltration centrifuge tube.
7. The method according to claim 1, wherein the final concentration of glycosidase F used in step (3) is 2-20U/. Mu.l.
8. The method according to claim 7, wherein the final concentration of glycosidase F used in step (3) is 4-10U/. Mu.l.
9. The method according to claim 1, wherein the organic solvent used for protein precipitation in step (4) is methanol, ethanol or acetone.
10. The method according to claim 9, wherein the organic solvent used for protein precipitation in step (4) is ethanol or acetone.
11. The process according to claim 1, wherein the volume ratio of the organic solvent used in the step (4) is 50% to 80%.
12. The process according to claim 11, wherein the organic solvent used in step (4) is present in an amount of 65% to 75% by volume.
13. The method according to claim 12, wherein the organic solvent used in the step (4) is 75% ethanol by volume.
14. Method according to any one of claims 1-13, characterized in that the method of evaluating the sialylation level of a glycoprotein oligosaccharide comprises the steps of:
(1) Carrying out denaturation and reduction treatment on the protein through guanidine hydrochloride and dithiothreitol, and carrying out alkylation treatment on the denatured and reduced protein;
(2) Changing liquid of the alkylated protein and the desalting column, collecting the liquid into a centrifuge tube, and concentrating the liquid to a certain volume;
(3) Carrying out enzyme digestion on the desalted and concentrated protein by using glycosidase F;
(4) Adding 75% ethanol by volume into the collected and concentrated protein, precipitating, centrifuging, and collecting supernatant to obtain oligosaccharide solution without protein;
(5) Concentrating and drying the oligosaccharide solution, and re-suspending to a certain volume to obtain an enriched oligosaccharide solution;
(6) Oligosaccharide profile detection analysis was performed on the enriched sugar chains using a high performance anion exchange chromatography-integrated pulse amperometric detector.
15. The method according to claim 14, characterized in that the method of high performance ion exchange chromatography detection is as follows:
adding 200 μl of ultrapure water into the dried oligosaccharide, completely dissolving, centrifuging at 13000rpm for 10min, taking supernatant, performing ion chromatography detection, wherein the ion chromatograph is a Thermo Scientific ICS-5000+DC system, the chromatographic column uses DionexCarboPac PA 200.2003×250mm, integrating different sialylated peaks after sample collection, and performing data analysis;
the ion chromatography detection instrument is set, mobile phase, gradient and other parameters as follows:
flow rate: 0.5ml/min
Column temperature: 30 DEG C
Sample injection volume: 25 μl full-loop sample injection
Mobile phase a:0.02M sodium hydroxide solution
Mobile phase B:0.1M sodium hydroxide solution
Mobile phase C:0.1M sodium hydroxide+1M sodium acetate solution
The ion chromatography elution gradient is as follows:
。
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