CN111961130A

CN111961130A - Method for extracting and separating IgM and IgG from blood plasma

Info

Publication number: CN111961130A
Application number: CN202010893241.9A
Authority: CN
Inventors: 肖岚; 刘余江; 张海梦; 任虹; 滕世超; 张宝献
Original assignee: Hualan Biological Engineering Co ltd; Hualan Gene Engineering Co ltd; HUALAN BIOLOGICAL ENGINEERING (CHONGQING) Inc
Current assignee: Hualan Biological Engineering Co ltd; Hualan Gene Engineering Co ltd; HUALAN BIOLOGICAL ENGINEERING (CHONGQING) Inc
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2020-11-20
Anticipated expiration: 2040-08-31
Also published as: CN111961130B

Abstract

The invention belongs to the field of protein extraction, and particularly discloses a method for extracting and separating IgM and IgG from blood plasma, wherein the pH value of the cold glue-removed blood plasma is adjusted to be 6.3-7.0, the conductivity of the cold glue-removed blood plasma is adjusted to be 9-12 mS/cm, and anion exchange chromatography is carried out to obtain a first flow-through liquid and an anion gel filler adsorbed with IgM protein; and adjusting the pH value of the first flow-through liquid to be 5.4-5.6, adjusting the conductivity of the first flow-through liquid to be 2.0-4.0 mS/cm, and performing cation exchange chromatography to obtain a second flow-through liquid and a cation gel filler adsorbed with IgG protein. By adopting the scheme of the invention, the problem of low IgM and IgG separation efficiency can be solved.

Description

Method for extracting and separating IgM and IgG from blood plasma

Technical Field

The invention belongs to the field of protein extraction, and particularly relates to a method for extracting and separating IgM and IgG from blood plasma.

Background

The immunoglobulin is a glycoprotein molecule produced by plasma cells under various physiological and pathological conditions through antigen stimulation, generally has a Y-shaped structure formed by two heavy chains and two light chains, and can be divided into five types of IgM, IgG, IgD, IgA and IgE, wherein the content of the IgG in blood plasma is 7-16 g/L and accounts for 75-80% of the total amount of the immunoglobulin; IgM has a very strong antigen neutralizing and immunoregulatory effect in antibody-mediated humoral immune reaction, and is an antibody which is first produced after an organism resists infection and is immune-stimulated.

The novel coronavirus pneumonia outbreaks since 12 months in 2019, and the diagnosis and treatment scheme issued by the national health and health commission refers to the basis of increasing serum detection on the basis of original nucleic acid detection and sequencing in a diagnosis and treatment standard, namely the detection of specific IgM and IgG antibodies of the novel coronavirus. Subsequent treatment of the novel coronary pneumonia also showed: specific antibodies, specific immunoglobulins and basic non-specific immunoglobulins are each effective in the treatment of novel coronaviruses. Research shows that specific antibody can neutralize and eliminate coronavirus while immunoglobulin (IgM and IgG) can inhibit cell factor storm and has clinical favorable effect.

In the prior art, a low-temperature ethanol precipitation method is generally adopted, namely, under a low-temperature environment, ethanol is added into cold glue-removed plasma for precipitation and filter pressing to obtain precipitates and suspension, a series of steps such as caprylic acid treatment and the like are carried out to obtain protein solution, and then a plurality of different proteins (including albumin, immunoglobulin and the like) are respectively extracted from the obtained protein solution. The low-temperature ethanol precipitation method aims at extracting a plurality of proteins in plasma as much as possible, so that a plurality of processing steps are carried out on the plasma, and the needed IgG protein (the yield is about 50%) can be separated from the plurality of proteins extracted subsequently, but the whole low-temperature ethanol precipitation method needs long time, the IgG separation efficiency is low, and the IgM protein is not separated independently.

In addition, the low-temperature ethanol precipitation method is generally carried out at 20 ℃ below zero, energy consumption is high, ethanol belongs to flammable and explosive substances, explosion-proof equipment needs to be arranged in a workshop of a factory due to safety considerations, and production cost is high.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a method for extracting and separating IgM and IgG from blood plasma, which aims to solve the problems that the low-temperature ethanol precipitation method is low in IgG separation efficiency, IgM protein is not separated independently, the separation is usually carried out at the temperature of minus 20 ℃, energy is consumed, ethanol is a flammable and explosive substance, and explosion-proof equipment needs to be arranged in a workshop of a factory building due to safety consideration, so that the production cost is high.

In order to achieve the purpose, the invention adopts the following technical scheme:

adjusting the pH value of the cold glue removed plasma to be 6.3-7.0, adjusting the conductivity of the cold glue removed plasma to be 9-12 mS/cm, and performing anion exchange chromatography to obtain a first flow-through liquid and an anion gel filler adsorbed with IgM protein; and adjusting the pH value of the first flow-through liquid to be 5.4-5.6, adjusting the conductivity of the first flow-through liquid to be 2.0-4.0 mS/cm, and performing cation exchange chromatography to obtain a second flow-through liquid and a cation gel filler adsorbed with IgG protein.

Compared with the prior art, the invention has the following beneficial effects:

1. the needed IgG and IgM can be directly extracted from the blood plasma by two times of ion exchange chromatography, the time of ethanol precipitation procedure treatment in the traditional extraction mode is reduced, and the extraction and separation efficiency is improved. And the method used in the scheme does not need to set a refrigerant to create a lower-temperature environment, and compared with the traditional low-temperature ethanol precipitation method, the method greatly reduces the energy consumption and further reduces the production cost.

2. Experiments prove that in the method used in the scheme, the pH and the conductivity during ion exchange chromatography have great influence on the extraction result, and the purity of the extracted IgG can reach 83 percent and the purity of the IgM can reach 75 percent within the pH and conductivity range used in the scheme.

Drawings

FIG. 1 is the TAME gel chromatography experimental spectrum.

FIG. 2 is a Unigel-80SP gel chromatography experimental chart.

In the figure: 1. a conductivity curve; 2. flow through the peak; 3. eluting peaks; 4. salting out peaks; 5. CIP peak.

Detailed Description

The invention is further described in detail below with reference to the accompanying drawings, and specific embodiments are given.

A method for extracting and separating IgM and IgG from blood plasma. The method is characterized by comprising the following steps:

s1, preparation of plasma

Thawing fresh frozen plasma, continuously centrifuging at 0-3 deg.C, removing cold glue to obtain supernatant, which is cold glue-removed plasma, and filtering with filter element with pore diameter of 0.22 μm to obtain clarified cold glue-removed plasma. Most of blood coagulation factors and lipoproteins are removed by centrifugal precipitation in a low-temperature environment, so that the required immunoglobulin can be better extracted in the subsequent steps.

S2 preparation for anion exchange chromatography

The chromatographic packing for anion exchange chromatography is strong anion packing, and TMAE anion gel packing is specifically selected in this embodiment.

Preparing a TMAE anion exchange packing chromatographic column: and (3) balancing the TMAE anion gel filler by using an equilibrium buffer solution containing 0.08M disodium hydrogen phosphate and citric acid, wherein the pH value of the equilibrium buffer solution is 6.3-7.0, the conductivity is 9-12 mS/cm, and the sample is treated to be loaded.

S3, anion exchange chromatography

Adjusting the plasma with cold removed gel obtained in step S1: the pH value is 6.3-7.0, the conductivity is 9-12 mS/cm, and the protein content is 45g/L +/-5 g/L, when in actual extraction, the pH and the conductivity of the equilibration buffer solution used in the step S2 are completely consistent with those of the cold-removed plasma in the step, for example, the pH of the cold-removed plasma in the step S2 is adjusted to 6.5, the pH of the equilibration buffer solution used in the step S2 is also 6.5, the conductivity of the cold-removed plasma in the step S2 is adjusted to 10mS/cm, and therefore the pH and the conductivity of the cold-removed plasma in the extraction process need to be determined in advance before extraction.

Filtering the adjusted cold glue-removed plasma by using a 0.22 mu m filter element, loading the TMAE anion exchange filler chromatographic column obtained in the step S2, performing anion exchange chromatography, collecting a first flow-through liquid in a linear mode of 1-3 cm/min, wherein a target protein containing IgM is combined in the TMAE anion gel filler, albumin, IgG protein and the like are flowed through, and collecting the first flow-through liquid for next step of IgG separation.

And (3) balancing 5CV of TMAE gel filler by using a balance buffer solution containing 0.08M disodium hydrogen phosphate and citric acid, wherein the pH value of the balance buffer solution is 6.3-7.0, the conductivity is 9-12 mS/cm, and the pH value and the conductivity of the balance buffer solution are completely consistent with those of the cold glue removing plasma in the step. And then phosphate eluent containing 0.08M disodium hydrogen phosphate and citric acid is used, the pH value of the phosphate eluent is 5.0, the conductivity is 10mS/cm, IgM protein adsorbed in the TAME anion gel filler is desorbed to obtain primary eluting collected liquid containing IgM, and subsequently, salt can be continuously used for washing off non-IgM protein bound in the TAME gel filler.

S4 cation exchange chromatography preparation

The chromatographic packing for cation exchange chromatography is strong cation packing, and in this example, Unigel-80SP cation gel packing is specifically selected.

Preparation of Unigel-80SP cation exchange packing chromatographic column: and (3) balancing the Unigel-80SP cationic gel filler by using an equilibrium buffer solution containing 0.025M disodium hydrogen phosphate and citric acid, wherein the pH value of the equilibrium buffer solution is 5.4-5.6, the conductivity is 2.0-4.0 mS/cm, and treating to obtain a sample.

S5 cation exchange chromatography

Adjusting the first flow-through obtained in step S3: the pH value is 5.4 to 5.6, the conductivity is 2.0 to 4.0mS/cm, and the protein content is 7g/L ± 1g/L, when actually extracting, the pH and the conductivity of the equilibration buffer solution used in step S4 need to be completely consistent with those of the first flow-through solution in this step, for example, if the pH of the first flow-through solution is adjusted to 5.5 in this step, the pH of the equilibration buffer solution used in step S4 also needs to be 5.5, if the conductivity of the first flow-through solution is adjusted to 3mS/cm in this step, the conductivity of the equilibration buffer solution used in step S4 also needs to be 3mS/cm, and therefore, the pH and the conductivity of the first flow-through solution in this extraction process need to be determined in advance before extracting.

And filtering the adjusted first flow-through liquid by using a 0.22 mu m filter element, loading the Unigel-80SP cation exchange filler chromatographic column obtained in the step S4, performing cation exchange chromatography, and collecting a second flow-through liquid at a linear flow rate of 1-3 cm/min, wherein IgG protein is combined in the Unigel-80SP cation gel filler, non-target proteins such as albumin and the like are flowed through, and the collected second flow-through liquid can be subsequently used for further purification and separation of the albumin.

Desorbing the IgG-containing protein from the Unigel-80SP cationic gel packing material by using a 0.15M sodium chloride +10mM sodium citrate solution to obtain a second elution collection liquid containing the IgG protein.

S6, subsequent processing

Subsequently, the IgM product can be purified from the first elution collected liquid obtained in step S3, and the IgG product can be purified from the second elution collected liquid obtained in step S5, which are not repeated herein.

The above protocol was tested:

1. experiment and detection equipment

The experimental equipment adopts GE Healthcare

awant150；

IgM, IgG and total protein content detection equipment adopts a Mirey biochemical analyzer.

2. Experimental result calculation method

The IgM and IgG purity calculation method comprises the following steps: respectively obtaining IgM and IgG purity values in the sample by respectively adopting the ratio of the IgM and IgG content in the sample to the total protein content;

IgM and IgG yield calculation method: obtaining the yield of IgM and IgG in samples in different steps according to the ratio of IgM and IgG content in samples in different experimental steps of chromatography (namely the IgM and IgG detection values are multiplied by the sample volume) to IgM and IgG content in samples before chromatography (namely the IgM and IgG detection values are multiplied by the sample collection volume);

the purity and yield are calculated according to data provided by experimental equipment, and certain errors exist.

3. Experiments 1 to 5 were carried out

Experiments 1-5 to examine IgM extractions, the main parameters were changed to those in step S3, as shown in the following table:

	experiment 1	Experiment 2	Experiment 3	Experiment 4	Experiment 5
						pH of plasma with cold removing gel	6.3±0.02	6.5±0.02	6.8±0.02	7.0±0.02	6.5±0.02
Conductivity of blood plasma with cold glue (mS/cm)	10±0.2	10±0.2	10±0.2	10±0.2	9±0.2

Except for the above parameters in step S3 (the parameter settings of the equilibration buffer in step S2 are identical to those of the plasma with no cryogel in step S3), the rest of the experimental steps are identical, and the purity and yield of IgM are determined according to the manner described above after the experiment, and the experimental results are shown in the following table (TAME gel chromatography experimental chart is shown in fig. 1, fig. 1 includes conductivity curve 1 and OD280 uv absorption curve, and OD280 uv absorption curve includes flow-through peak 2, elution peak 3, salting-out peak 4, CIP peak 5):

	experiment 1	Experiment 2	Experiment 3	Experiment 4	Experiment 5
						Purity of IgM	56％	74％	67％	44％	75％
Yield of IgM	30％	77％	64％	55％	72％

Conclusion of experiments 1-5:

according to experiments 1-4, within the pH range of 6.50-6.80, the yield of IgM can reach more than 60%, the purity can reach about 70%, and higher or lower pH value has great influence on both the yield and the purity of the product. According to

experiments

2 and 5, the influence of the conductivity on the extraction result is small, and in the actual extraction process, the data of experiment 2 can be optimized, namely the pH of the cold glue removing blood plasma is adjusted to be 6.5, and the conductivity is adjusted to be 10 mS/cm.

4. Experiments 6 to 13 were carried out

Experiments 6-13 tested the extraction of IgG with the main parameters changed to those in step S5, as shown in the following table:

except for the difference in the above parameters in step S5 (the parameter settings of the equilibration buffer in step S4 are identical to the first flow-through in step S5), the rest of the experimental steps are completely identical, and the IgG purity and IgG yield are measured after the experiment, and the experimental results are shown in the following table (UniGel-80SP gel chromatography experimental chart is shown in fig. 2, fig. 2 includes conductivity curve 1 and OD280 uv absorption curve, and OD280 uv absorption curve includes flow-through peak 2 and elution peak 3):

experiment 6

Experiment 7

Experiment 8

Experiment 9

Experiment 10

Experiment 11

Experiment 12

Experiment 13

IgG purity

63％

83％

76％

82％

67％

76％

72％

76％

IgG yield

91％

98％

95％

100％

101％

87％

99％

89％

Experiment 6-13 conclusions:

according to experiments 8, 11 and 13, on the premise of the same conductivity, the IgG yield is higher and reaches more than 90% under the condition of pH5.50. According to experiments 6-9, on the premise of the same pH (5.50), the IgG purity is found to be greatly different due to the change of the conductivity, but the IgG yield and the purity can also meet the expected requirements, the yield can be ensured to be about 90%, and in the actual extraction process, in order to ensure that the IgG purity can be controlled to be more than 70%, the pH of the first flow-through liquid is preferably adjusted to be 5.50, and the conductivity is 3.0-4.0 mS/cm.

The experiment shows that in the method for separating IgM and IgG by the method, the yield of IgG reaches more than 90%, the purity of IgG reaches 83%, the yield of IgM reaches more than 60%, and the purity of IgM reaches 75%.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. A method for extracting and separating IgM and IgG from plasma, which comprises: adjusting the pH value of the cold glue removed plasma to be 6.3-7.0, adjusting the conductivity of the cold glue removed plasma to be 9-12 mS/cm, and performing anion exchange chromatography to obtain a first flow-through liquid and an anion gel filler adsorbed with IgM protein;

and adjusting the pH value of the first flow-through liquid to be 5.4-5.6, adjusting the conductivity of the first flow-through liquid to be 2.0-4.0 mS/cm, and performing cation exchange chromatography to obtain a second flow-through liquid and a cation gel filler adsorbed with IgG protein.

2. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: the preparation method of the blood plasma with the cold glue removed comprises the following steps: after the frozen plasma is melted, continuously centrifuging at the temperature of 0-3 ℃, removing cold glue, and obtaining supernatant fluid which is the cold glue removed plasma.

3. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: adjusting pH of the cold glue removed plasma to 6.5, and conductivity to 10 mS/cm; adjusting the pH value of the first flow-through liquid to 5.5 and the conductivity to 3.0-4.0 mS/cm.

4. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: during the anion exchange chromatography, the anion gel filler is balanced by phosphate-containing equilibrium buffer solution, and the pH and the conductivity of the equilibrium buffer solution are consistent with those of the plasma without the cold gel.

5. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: and desorbing the IgM protein adsorbed in the anion gel filler by using a phosphate eluent with the pH value of 5.0 and the conductivity of 10mS/cm after the anion exchange chromatography to obtain a first eluting and collecting liquid containing IgM.

6. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: and during the cation exchange chromatography, the cation gel packing is balanced by phosphate-containing balance buffer solution, and the pH and the conductivity of the balance buffer solution are consistent with those of the first flow-through solution.

7. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: after the cation exchange chromatography, the IgG protein adsorbed in the cation gel filler is desorbed by a mixed solution of 0.15M sodium chloride and 10mM sodium citrate to obtain a second elution collection liquid containing IgG.

8. The method of claim 4 or 6, wherein the IgM and IgG are extracted and separated from plasma by: the equilibrium buffer solution is a mixed solution of disodium hydrogen phosphate and citric acid.

9. The method of claim 5, wherein the IgM and IgG compounds are extracted and separated from plasma by: the phosphate eluent is a mixed solution of disodium hydrogen phosphate and citric acid.

10. The method of claim 1 for the extraction and separation of IgM and IgG from plasma, wherein: the chromatographic packing for cation exchange chromatography is strong cation packing, and the chromatographic packing for anion exchange chromatography is strong anion packing.