CN114460186A - High performance liquid chromatography for analyzing sialic acid content in sample - Google Patents
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 48
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 title claims abstract description 27
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 105
- 238000000034 method Methods 0.000 claims abstract description 24
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 21
- 238000002156 mixing Methods 0.000 claims abstract description 8
- 239000011259 mixed solution Substances 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000004811 liquid chromatography Methods 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims description 4
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 claims description 4
- 238000000108 ultra-filtration Methods 0.000 claims description 4
- SQVRNKJHWKZAKO-LUWBGTNYSA-N N-acetylneuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-LUWBGTNYSA-N 0.000 claims description 2
- 229960002964 adalimumab Drugs 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 3
- 238000002798 spectrophotometry method Methods 0.000 abstract description 3
- 238000004949 mass spectrometry Methods 0.000 abstract description 2
- SQVRNKJHWKZAKO-PFQGKNLYSA-N N-acetyl-beta-neuraminic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-PFQGKNLYSA-N 0.000 description 19
- FDJKUWYYUZCUJX-UHFFFAOYSA-N N-glycolyl-beta-neuraminic acid Natural products OCC(O)C(O)C1OC(O)(C(O)=O)CC(O)C1NC(=O)CO FDJKUWYYUZCUJX-UHFFFAOYSA-N 0.000 description 17
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 16
- 238000010306 acid treatment Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 5
- 229940125644 antibody drug Drugs 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001212 derivatisation Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- SUHQNCLNRUAGOO-UHFFFAOYSA-N N-glycoloyl-neuraminic acid Natural products OCC(O)C(O)C(O)C(NC(=O)CO)C(O)CC(=O)C(O)=O SUHQNCLNRUAGOO-UHFFFAOYSA-N 0.000 description 1
- FDJKUWYYUZCUJX-KVNVFURPSA-N N-glycolylneuraminic acid Chemical compound OC[C@H](O)[C@H](O)[C@@H]1O[C@](O)(C(O)=O)C[C@H](O)[C@H]1NC(=O)CO FDJKUWYYUZCUJX-KVNVFURPSA-N 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 102000014105 Semaphorin Human genes 0.000 description 1
- 108050003978 Semaphorin Proteins 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 229940049595 antibody-drug conjugate Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Life Sciences & Earth Sciences (AREA)
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Abstract
The invention discloses a high performance liquid chromatography method for analyzing sialic acid content in a sample, which comprises the step of mixing acetic acid with a protein sample to obtain a mixed solution so as to release sialic acid in the protein sample. The invention solves the problems of weak specificity of a spectrophotometry method, poor repeatability of a thin layer method, high cost of a mass spectrometry method and the like in the prior analysis technology, thereby providing a simple high performance liquid chromatography with strong specificity and high accuracy for analyzing sialic acid content in different types of antibodies.
Description
Technical Field
The invention relates to the field of chemical substance detection, in particular to a high performance liquid chromatography for analyzing sialic acid content in a sample.
Background
New theories and new technologies of novel engineering antibodies such as bispecific antibodies, antibody drug conjugates, glycosyl engineering antibodies, nano antibodies and the like are continuously emerging. Most antibody drugs are expressed by mammalian cells, and the correct folding, spatial conformation and modification of the translated gene determine the normal function of the antibody drug. Therefore, controlling the type and number of post-translationally modified variants is one of the great challenges facing the development process of antibody drugs. The mass of the sugar chain in the antibody drug is 2% of the whole antibody molecule, and for the monoclonal antibody for treating autoimmune diseases, the number of sialic acid modifications at the end of the sugar chain may have a strong positive correlation with its anti-inflammatory effect. Most common is N-acetylneuraminic acid, and the sialic acid is physically analyzed by a plurality of methods, such as spectrophotometry, thin-layer chromatography, high performance liquid chromatography, nuclear magnetic resonance, high performance liquid chromatography-mass spectrometry and the like. However, the spectrophotometry is not very specific, the thin layer method is poor in reproducibility, and the mass spectrometry is costly.
At present, the prior art establishes an analytical method (https:// www.instrument.com.cn/application/Solution-895694.html) for measuring the contents of sialic acid N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) in an antibody by using Shimadzu ultra high performance liquid chromatograph LC-30A, for example. However, trifluoroacetic acid used in the technology has the phenomenon of insufficient acidolysis or the phenomenon of influencing derivatization reaction, and the subsequent detection result is not accurate enough.
Therefore, it is urgently needed to research a simple, highly specific and highly accurate high performance liquid chromatography for analyzing sialic acid content in different types of antibodies.
Disclosure of Invention
The invention aims to solve the technical problems of weak specificity, poor repeatability, high cost and the like in the existing analysis technology, thereby providing a high performance liquid chromatography for analyzing the sialic acid content in a sample.
In order to solve the above technical problems, the present invention provides a method for determining sialic acid in a protein sample, comprising the steps of:
A) mixing acetic acid with the protein sample to obtain a mixed solution, so that sialic acid in the protein sample is released; preferably, the method further comprises:
B) and incubating the mixed solution to obtain an incubation solution.
In a specific embodiment, the concentration of acetic acid is 3-5M, the ratio of acetic acid to the protein sample is 1:1, the incubation temperature is 80 ℃, and the protein sample is derived from adalimumab, such as, for example, tamethol.
In a particular embodiment, the acetic acid is at a concentration of 4M; the sialic acid is Neu5Ac and/or Neu5 Gc.
In a specific embodiment, before the mixing, the method further comprises:
(1) the protein sample is subjected to a liquid change treatment using a buffer solution such as PBS, for example, for 4 times, using an ultrafiltration tube, and a liquid-changed protein sample is obtained.
In a specific embodiment, after the incubating, the method further comprises:
C) adding DMB solution, mixing uniformly, and reacting for 2.5 h;
in a specific embodiment, the concentration of the DMB solution is 13mM, the ratio of the incubation liquid to the DMB is 1:1, and the reaction in C) is performed under conditions of light shielding and a temperature of 50 ℃.
In one embodiment, the DMB solution is DMB containing 1.5M acetic acid, 14mM sodium dithionite, 0.8M 2-mercaptoethanol, 13mM DMB.
In a specific embodiment, the method uses high performance liquid chromatography for detection, and the type of a liquid chromatography column used in the high performance liquid chromatography is Thermo Hypersil GoldTM aQ 250.
In a specific embodiment, the liquid chromatography column has an inner diameter of 4.6 mm.
In a specific embodiment, the mobile phase used in the liquid chromatography column is 7% methanol, 8% acetonitrile in water.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention adopts the optimal acidolysis reagent type, and solves the problem of inaccurate result caused by insufficient acidolysis degree of the sample. The Neu5Ac and Neu5Gc measured by the method have good linear relation in the concentration ranges of 0.0002-0.01 g/L and 0.0002-0.01 g/L respectively, the linear correlation coefficients are all larger than 0.999, the accuracy ranges are between 102% -106% and 101% -111%, the phenomenon of insufficient acidolysis or the phenomenon of influencing derivatization reaction is solved, and the accuracy is better.
Drawings
FIG. 1 is a Neu5Ac versus Neu5Gc standard curve; fig. 1 a is a standard curve of Neu5Gc at 4M acetic acid concentration, and fig. 1B is a standard curve of Neu5Ac at 4M acetic acid concentration.
FIG. 2 is a standard overlay of the concentration of Neu5Ac versus Neu5Gc under acetic acid.
Figure 3 is a semaphorin result map.
FIG. 4 shows the standard curves of Neu5Ac and Neu5Gc under different concentrations (2-6M) of acetic acid.
FIG. 5 is a standard overlay of the concentration of Neu5Ac versus Neu5Gc under trifluoroacetic acid conditions.
Detailed Description
For a more detailed understanding of the technical content, characteristics and effects of the present invention, the technical solutions of the present invention will now be described in further detail with reference to specific embodiments, but the present invention is not limited to the scope of the embodiments. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
The data in the following examples are obtained by averaging two parallel experiments.
Example 1
In the embodiment, the content of sialic acid in the crude medicines of the crude medicines is analyzed by the high performance liquid chromatography, the sialic acid is released by treating with acetic acid, and then the crude medicines are labeled by a derivatization reagent, and the specific operation steps are as follows:
step 1), taking 400 mu g of sample into a centrifuge tube, and adding PBS to make up to 400 mu L. After mixing, the mixture is transferred to an ultrafiltration tube and centrifuged for 10min at 13000rpm and 8 +/-2 ℃. After centrifugation, the volume is complemented to 400 mu L with PBS, the mixture is evenly mixed, centrifuged for 10min at 13000rpm and 8 +/-2 ℃, the step is repeated for 4 times, PBS is added to complement the volume to 100 mu L, and the trapped fluid in the ultrafiltration tube is transferred to a new centrifuge tube. After mixing, the protein concentration was determined using Nanodrop.
Step 2), add 25 μ L of 4M acetic acid to 25 μ L of 1mg/mL standard (Neu5Ac and Neu5 Gc)/protein sample (sumile), mix well and incubate for 2 hours at 80 ℃.
Step 3), add 50 μ L DMB solution (1.5M acetic acid, 14mM sodium dithionite, 0.8M 2-mercaptoethanol, 13mM DMB) into the above system, mix well, incubate for 2.5h at 50 ℃ in the dark. Cooling to room temperature and bottling for testing.
Step 4), preparing a mobile phase of 7% methanol and 8% acetonitrile water, and installing a chromatographic column Thermo Hypersil GoldTM aQ 250 x 4.6 mm.
Step 5), setting instrument parameters: excitation wavelength 373 nm; emission wavelength 448 nm; the column temperature is 35 +/-2 ℃; the temperature of the sample chamber is 8 +/-2 ℃; the injection volume is 20 mu L; the operation mode adopts an isocratic mode; the flow rate is 1.2 mL/min; run time 15 minutes.
Step 6), loading the sample prepared in the step 3) into an automatic sample injector
Step 7), after the results are processed, calculating the sialic acid content by using a formula:
TABLE 1 content of sialic acid by HPLC
| Product(s) | Neu5Gc (mol/mol protein) | Neu5Ac (mol/mol protein) |
| Xiumeile | ≤0.092 | ≤0.097 |
As a result, as shown in fig. 3, when the sample and the sialic acid standard were subjected to the same pretreatment and chromatograms were compared, it was found that the retention time of Neu5Gc and Neu5Ac in the sample was consistent with that of the standard, and the peak area of the sample was substituted into the Neu5Gc standard curve:
Y＝9.93×1010X-6.97×105
and Neu5Ac standard curve:
Y＝9.93×1010X-5.07×105
the sialic acid concentration in the sample was calculated and substituted into the sialic acid content calculation formula to obtain the results in table 1. In the present example, it can be seen from the combination of FIG. 3 and Table 1 that sialic acid content in the mAb product, Xiamelete, was successfully obtained.
Comparative example 1 acid liquor comparison of different concentrations
Comparing R of standard curves of Neu5Gc and Neu5Ac under different concentrations (2-6M) of acetic acid2，R2Closer to 1, the more linear the sialic acid standard is represented by the corresponding acetic acid concentration.
According to the preparation steps in example 1, the protein samples after liquid change are mixed with acetic acid with the concentrations of 2M, 3M, 4M, 5M and 6M respectively, incubated, and detected according to the following method:
step 1), add 25. mu.L of 2M, 3M, 4M, 5M, 6M acetic acid to 25. mu.L of 1mg/mL standard, mix well, incubate for 2 hours at 80 ℃.
Step 2), 50. mu.L of DMB solution (1.5M acetic acid, 14mM sodium dithionite, 0.8M 2-mercaptoethanol, 13mM DMB) was added to the above system, mixed well, and incubated at 50 ℃ for 2.5 hours in the dark. Cooling to room temperature and bottling for testing.
Step 3), preparing a mobile phase of 7% methanol and 8% acetonitrile water, and installing a chromatographic column Thermo Hypersil GoldTM aQ 250 x 4.6 mm.
Step 4), setting instrument parameters: excitation wavelength 373 nm; emission wavelength 448 nm; the column temperature is 35 +/-2 ℃; the temperature of the sample chamber is 8 +/-2 ℃; the injection volume is 20 mu L; the operation mode adopts an isocratic mode; the flow rate is 1.2 mL/min; run time 15 minutes.
And 5) loading the sample prepared in the step 4) into an automatic sample injector.
According to the results shown in FIGS. 1 (A of FIG. 1 and B of FIG. 1) and 4 (A of FIG. 4-H of FIG. 4), R of the standard curves of Neu5Gc and Neu5Ac was observed under acetic acid at a concentration of 3 to 5M2Both are close to 1, but R of the standard curves of Neu5Gc and Neu5Ac under 2M acetic acid concentration20.977 and 0.975, respectively, less than 0.99; r of the standard curve of Neu5Gc versus Neu5Ac at 6M acetic acid concentration20.977 and 0.974, respectively, less than 0.99. It can be seen that too high or too low an acetic acid concentration has an effect on the linearity of the standard, and therefore the optimum treatment conditions are 4M acetic acid, R of the standard curve for Neu5Gc versus Neu5Ac2Are all 1.00.
Comparative example 2 comparison of acid solutions
The acetic acid in step (2) of example 1 was replaced with trifluoroacetic acid, and the other conditions were the same as in example 1, comparing the difference between trifluoroacetic acid and acetic acid.
The results are shown in tables 2 and 3.
TABLE 2 recovery under trifluoroacetic acid treatment conditions
*: because the concentration of the sample is diluted by one time during detection, about half of the actually measured concentration which is the theoretical standard-added concentration is a normal value.
TABLE 3 recovery under acetic acid treatment
*: because the concentration of the sample is diluted by one time during detection, about half of the actually measured concentration which is the theoretical standard-added concentration is a normal value.
As shown in FIGS. 2 and 5, under the 4M acetic acid treatment condition, the two kinds of sialic acid standards, Neu5Gc and Neu5Ac, have good linearity in the concentration ranges of 0.0002-0.01 g/L and 0.0002-0.01 g/L, respectively, and are superior to trifluoroacetic acid.
As can be seen from tables 2 and 3, under the conditions of 4M acetic acid and 4M trifluoroacetic acid treatment, different concentrations of standards were added to the same sample, and then analyzed to yield the recovery of the added different concentrations of standards. The results are shown in the table above and the comparison shows that the average recovery is 111% under trifluoroacetic acid treatment and 105% under acetic acid treatment. Therefore, the acetic acid treatment condition can obtain a better accuracy result, and the accuracy of the detection result is improved.
Claims (10)
1. A method for determining sialic acid in a protein sample, comprising the steps of:
A) mixing acetic acid with the protein sample to obtain a mixed solution, so that sialic acid in the protein sample is released; preferably, the method further comprises:
B) and incubating the mixed solution to obtain an incubation solution.
2. The method of claim 1, wherein the concentration of acetic acid is 3-5M, the ratio of acetic acid to the protein sample is 1:1, the temperature of the incubation is 80 ℃, and the protein sample is from adalimumab such as sumatriol.
3. The method of claim 2, wherein the acetic acid is at a concentration of 4M; the sialic acid is Neu5Ac and/or Neu5 Gc.
4. The method of claim 1, further comprising, prior to the mixing:
(1) the protein sample is subjected to a liquid change treatment using a buffer solution such as PBS, for example, for 4 times, using an ultrafiltration tube, and a liquid-changed protein sample is obtained.
5. The method of claim 1, further comprising, after said incubating:
C) DMB solution is added and mixed well, and reacted for 2.5 h.
6. The method according to any one of claims 1 to 5, wherein the concentration of the DMB solution is 13mM, the ratio of the incubation solution to the DMB is 1:1, and the reaction in C) is carried out under conditions protected from light and at a temperature of 50 ℃.
7. The method of claim 6, wherein the DMB solution is DMB solution containing 1.5M acetic acid, 14mM sodium dithionite, 0.8M 2-mercaptoethanol, 13mM DMB.
8. The method of claim 1, wherein the detection is performed using high performance liquid chromatography using a liquid chromatography column model of Thermo Hypersil GoldTM aQ 250.
9. The method of claim 8, wherein the liquid chromatography column has an inner diameter of 4.6 mm.
10. The method of claim 8, wherein the liquid chromatography column uses a mobile phase of 7% methanol, 8% acetonitrile in water.
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