CN109111498A - A method of it reducing albumen and forms polymer in protein L gel chromatography - Google Patents
A method of it reducing albumen and forms polymer in protein L gel chromatography Download PDFInfo
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- CN109111498A CN109111498A CN201811061562.1A CN201811061562A CN109111498A CN 109111498 A CN109111498 A CN 109111498A CN 201811061562 A CN201811061562 A CN 201811061562A CN 109111498 A CN109111498 A CN 109111498A
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- China
- Prior art keywords
- protein
- eluent system
- gel chromatography
- albumen
- eluent
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
Abstract
The present invention relates to a kind of reduction albumen methods for forming polymer in protein L gel chromatography, comprising: (1) in protein L eluent system addition cysteine (Cys), using in NaOH solution and eluent system；(2) sample after albumen protein L gel chromatography is handled using G25 sephadex chromatography, for removing Cys.Polymer caused by the present invention effectively inhibits low pH to elute is formed, and there is no degradations for purifying protein.Present invention process is easy to operate, and effect is obvious, provides reference for protein L gel chromatography technique in laboratory research and industrial production, also provides new approaches further to study the method that the other albuminoids of removal form polymer in purifying.
Description
Technical field
The present invention relates to a kind of methods that reduction albumen forms polymer in protein L gel chromatography, belong to biology
Technology technical field.
Background technique
Protein L is a kind of affinity chromatography medium (resin) captured suitable for antibody and antibody fragment, can be in not shadow
In the case where sound antigen binding in conjunction with Kappa (κ) light chain of immune globulin bletilla immunoglobulin, there is broader antibody
Piece Selection range, higher purity and yield.But the optimum washing engaging condition pH value of its mesh albumen is lower, general pH value is 3
Even lower condition, high concentration albumen influence albumen after chromatography in, easy to form polymer extremely unstable compared under low ph conditions
Molecular arrangement and active recovery efficiency.
Recombinant protein is that a kind of recombinated with the high activity that people's epidermal growth factor receptor (HER2) is identification target spot merges egg
It is white, it is a kind of novel targeted anti-breast cancer protein drug.With simple production process, bioactivity is high, and it is excellent that impurity in products is few etc.
Point has good application value and market prospects.During using protein L column chromatography, because elution requirement restricts
And form a certain amount of polymer.The formation of polymer and the number of content directly affect the quality of product, clinical application
The qualification rate of safety and product.From medical safety, the content for reducing or removing as far as possible polymer is very important
One of process procedure.Current study show that this method can significantly inhibit recombinant protein on the basis of improving fusion protein purity
The formation of polymer reaches certain mass index request.
Summary of the invention
The purpose of the present invention is to provide the sides that a kind of reduction albumen forms polymer in protein L gel chromatography
Method, the present invention add the reagents such as cysteine by chromatographing to protein L, solve in purifying in the elution buffer used
The problem of polymer formation.So as to improve lipidated protein and purification efficiency, and generated to study in other albuminoid purifying
The removal of polymer provides reference.Technical scheme is as follows:
A method of it reducing albumen and forms polymer in protein L gel chromatography, including
(1) cysteine (Cys) is added in protein L eluent system, using in NaOH solution and eluent system；
(2) sample after albumen protein L gel chromatography is handled using G25 sephadex chromatography, for going
Except Cys；
The pH of eluent system is 2.0-7.0 in the step (1)；Preferably, eluent system is selected from citrate buffer solution, vinegar
Acid buffer, glycine-HCI buffer, phthalic acid-hydrochloride buffer, disodium hydrogen phosphate-citrate buffer solution, lemon
Any one in acid-sodium citrate buffer solution, acetic acid-sodium acetate buffer solution.
Fusion protein 4D5Fv-PE25 includes variable region Fv and Pseudomonas Exotoxin PE25 (the invention name of Herceptin
Referred to as fusion protein 4D5Fv-PE25 and its preparation method and application, application No. is 201510674652.8)
Further, the Cys of 0.1M is added in eluent system, using in the NaOH solution of 0.5M and eluent system is to pH
It is 8.5 ± 0.3.
Further, the side that fusion protein 4D5Fv-PE25 forms polymer in protein L gel chromatography is reduced
Method, the specific steps are as follows:
(1) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes；
(2) fusion protein 4D5Fv-PE25 is subjected to loading with the applied sample amount of 20mg/ml gel；
(3) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes again；
(4) albumen wash-out is carried out with the eluent system containing cysteine, and collects eluent；
(5) after slightly mixing eluent system, it is 8.5 ± 0.3 that eluent system, which is neutralized to pH, using NaOH solution immediately；
(6) neutralizer is concentrated into protein content using the method for ultrafiltration concentration is 2mg/ml；
(7) it by after concentrating sample loading to the G25 sephadex column balanced with PBS, is eluted, is received with PBS buffer solution
Collection eluent can be obtained the monomer of destination protein.
Further, albumen wash-out is carried out with the eluent system containing 0.1M cysteine in the step (4)；It is preferred that
, albumen wash-out is carried out with the 30mM citric acid elution buffer containing 0.1M cysteine.
It further, immediately will using the NaOH solution of 0.5M after slightly mixing eluent system in the step (4)
It is 8.5 ± 0.3 that eluent system, which is neutralized to pH,.
Compared with the prior art, the present invention has the following advantages:
Cys is added in protein L eluent and by the method for sum in NaOH solution by the present invention, effectively inhibits low
Polymer caused by pH is eluted is formed.Cys, which is added in eluent system, does not influence original elution requirement, while G25 glucan is solidifying
Cys can be efficiently removed when glue-line analysis replacement buffer.Using in NaOH solution and eluent, there is no drops for purifying protein
It solves, Cys can not only effectively reduce the formation of polymer in purification process in the present invention, moreover it is possible to prevent from recombinating in N-process
Protein degradation is a kind of excellent protective agent.The present invention can effectively inhibit the polymer formed by protein purification, technological operation letter
Single, effect is obvious, provides reference for protein L gel chromatography technique in laboratory research and industrial production, is also further
Research removes the method that other albuminoids form polymer in purifying and provides new approaches.
Detailed description of the invention
To be illustrated more clearly that background technique or technical solution of the present invention, below to the prior art or specific embodiment
The attached drawing of middle combined use is briefly described；It should be evident that below in conjunction with the attached drawing side of being only for of specific embodiment
Just understand the embodiment of the present invention, for those of ordinary skill in the art, without creative efforts, may be used also
To obtain other drawings based on these drawings；
Fig. 1 is to elute effect picture using different eluent systems after protein L gel chromatography in embodiment 1；
Fig. 2 is to elute effect picture using different eluent systems after protein L gel chromatography in embodiment 2；
Fig. 3 is to elute effect picture using different eluent systems after protein L gel chromatography in embodiment 3；
Fig. 4 is to elute effect picture using different eluent systems after protein L gel chromatography in embodiment 4；
Fig. 5 is to elute effect picture using different eluent systems after protein L gel chromatography in embodiment 5；The peak CIP: former
Position cleaning peak；
Fig. 6 is Content of polymer detection figure；Wherein, 1 be eluent system is to be added in 30mM citric acid elution buffer
25mM Cys；2 be eluent system be in 30mM citric acid elution buffer addition 50mM Cys；3 be eluent system be 30mM lemon
100mM Cys is added in lemon acid elution buffer.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art answer
It should be appreciated that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form repair
Change or replace, but these modifications and replacement are fallen within the protection scope of the present invention.
A kind of method for reducing albumen and forming polymer in protein L gel chromatography of embodiment
(1) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes, flow velocity 1.0ml/min detects wave
Long 280nm；
(2) fusion protein 4D5Fv-PE25 is subjected to loading with the applied sample amount of 20mg/ml gel；
(3) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes；
(4) albumen wash-out is carried out with eluent system, and collects eluent；
(5) after slightly mixing eluent system, it is 8.5 that eluent system, which is neutralized to pH, using the NaOH solution of 0.5M immediately
±0.3；
(6) neutralizer is concentrated into protein content using the method for ultrafiltration concentration is 2mg/ml；
(7) it by after concentrating sample loading to the G25 sephadex column balanced with PBS, is eluted, is received with PBS buffer solution
Collection eluent can be obtained the monomer of destination protein.
Eluent system is shown in Table 1:
Table 1
Test example 1 utilizes protein L gel chromatography, different eluent system Protein Separation situations
Through detecting, there is good separating effect, as shown in Figure 1, embodiment 2, embodiment 3 and embodiment 5 in embodiment 1
In be only capable of elution lower part and divide destination protein, lower destination protein could be washed when needing to clean chromatographic column completely, respectively such as Fig. 2, Fig. 3 and
Map baseline is only caused to increase shown in Fig. 5, in embodiment 4, elution efficiency is unaffected, as shown in Figure 4.
The detection of 2 Content of polymer of test example
Using in NaOH in reference examples 1, reference examples 2 and embodiment 4 after being handled using non-reduced SDS-PAGE electrophoresis detection
With rear Content of polymer
Reference examples 1: eluent system is that 25mM Cys, the same embodiment of other steps are added in 30mM citric acid elution buffer
4；
Reference examples 2: eluent system is that 50mM Cys, the same embodiment of other steps are added in 30mM citric acid elution buffer
4。
Testing result is as shown in fig. 6,1 be eluent system be that 25mM is added in 30mM citric acid elution buffer in Fig. 6
Cys；2 be eluent system be in 30mM citric acid elution buffer addition 50mM Cys；3 be eluent system be 30mM lemon pickling
100mM Cys (embodiment 4) is added in de- buffer；It can be seen that with Cys concentration increase Content of polymer in eluent system
It gradually decreases, polymer is not generally visible when Cys concentration is improved to 100mM.
Claims (8)
1. a kind of method for reducing albumen and forming polymer in protein L gel chromatography, which is characterized in that the method packet
It includes:
(1) cysteine (Cys) is added in protein L eluent system, using in NaOH solution and eluent system；
(2) sample after albumen protein L gel chromatography is handled using G25 sephadex chromatography, for removing
Cys；
The pH of eluent system is 2.0-7.0 in the step (1).
2. the method according to claim 1, wherein the eluent system is selected from citrate buffer solution, acetic acid delays
Fliud flushing, glycine-HCI buffer, phthalic acid-hydrochloride buffer, disodium hydrogen phosphate-citrate buffer solution, citric acid-
Any one in sodium citrate buffer solution, acetic acid-sodium acetate buffer solution.
3. utilizing 0.5M's the method according to claim 1, wherein the Cys of 0.1M is added in eluent system
In NaOH solution and eluent system to pH be 8.5 ± 0.3.
4. the method according to claim 1, wherein it is solidifying in protein L to reduce fusion protein 4D5Fv-PE25
The method of polymer is formed in glue-line analysis.
5. according to the method described in claim 4, it is characterized in that, specific step is as follows:
(1) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes；
(2) fusion protein 4D5Fv-PE25 is subjected to loading with the applied sample amount of 20mg/ml gel；
(3) protein L gel chromatography column is balanced with the PBS equilibrium liquid of 5 times of column volumes again；
(4) albumen wash-out is carried out with the eluent system containing cysteine, and collects eluent；
(5) after slightly mixing eluent system, it is 8.5 ± 0.3 that eluent system, which is neutralized to pH, using NaOH solution immediately；
(6) neutralizer is concentrated into protein content using the method for ultrafiltration concentration is 2mg/ml；
(7) it by after concentrating sample loading to the G25 sephadex column balanced with PBS, is eluted with PBS buffer solution, collection is washed
De- liquid can be obtained the monomer of destination protein.
6. according to the method described in claim 4, it is characterized in that, with washing containing 0.1M cysteine in the step (4)
Lift-off system carries out albumen wash-out.
7. according to the method described in claim 6, it is characterized in that, with containing 0.1M cysteine in the step (4)
30mM citric acid elution buffer carries out albumen wash-out.
8. according to the method described in claim 4, it is characterized in that, being stood after slightly mixing eluent system in the step (4)
It is 8.5 ± 0.3 that eluent system, which is neutralized to pH, using the NaOH solution of 0.5M.
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| CN201811061562.1A CN109111498A (en) | 2018-09-12 | 2018-09-12 | A method of it reducing albumen and forms polymer in protein L gel chromatography |
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| CN201811061562.1A CN109111498A (en) | 2018-09-12 | 2018-09-12 | A method of it reducing albumen and forms polymer in protein L gel chromatography |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521015A (en) * | 2020-10-21 | 2021-10-22 | 山东省妇幼保健院 | Fusion protein 4D5Fv-PE25 freeze-dried agent and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1406247A (en) * | 2000-10-24 | 2003-03-26 | 财团法人化学及血清疗法研究所 | Method of monomerizing human serum albumin polymers |
| CN106589131A (en) * | 2015-10-19 | 2017-04-26 | 山东省生物制品研究所 | Fusion protein 4D5Fv-PE25, preparation method and uses thereof |
| CN108085358A (en) * | 2018-02-08 | 2018-05-29 | 保定冀中药业有限公司 | A kind of preparation method of canine parvovirus monoclonal antibody IgG2b-Fab segments |
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2018
- 2018-09-12 CN CN201811061562.1A patent/CN109111498A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1406247A (en) * | 2000-10-24 | 2003-03-26 | 财团法人化学及血清疗法研究所 | Method of monomerizing human serum albumin polymers |
| CN106589131A (en) * | 2015-10-19 | 2017-04-26 | 山东省生物制品研究所 | Fusion protein 4D5Fv-PE25, preparation method and uses thereof |
| CN108085358A (en) * | 2018-02-08 | 2018-05-29 | 保定冀中药业有限公司 | A kind of preparation method of canine parvovirus monoclonal antibody IgG2b-Fab segments |
Non-Patent Citations (1)
| Title |
|---|
| 展波等: "减少重组蛋白TP25在Protein L层析中形成多聚体方法的建立", 《中国生物制品学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113521015A (en) * | 2020-10-21 | 2021-10-22 | 山东省妇幼保健院 | Fusion protein 4D5Fv-PE25 freeze-dried agent and preparation method and application thereof |
| CN113521015B (en) * | 2020-10-21 | 2022-12-06 | 山东省妇幼保健院 | Fusion protein 4D5Fv-PE25 freeze-dried agent and preparation method and application thereof |
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Address after: 250102 No. 238 Jingshidong Road, Lixia District, Jinan City, Shandong Province Applicant after: Shandong maternal and child health care hospital Address before: 271000 No. 14 Hudong Road, Taishan District, Taian City, Shandong Province Applicant before: Shandong Institute of Biological Products |