CN107970899A - A kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying - Google Patents
A kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying Download PDFInfo
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- CN107970899A CN107970899A CN201711212402.8A CN201711212402A CN107970899A CN 107970899 A CN107970899 A CN 107970899A CN 201711212402 A CN201711212402 A CN 201711212402A CN 107970899 A CN107970899 A CN 107970899A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/261—Synthetic macromolecular compounds obtained by reactions only involving carbon to carbon unsaturated bonds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
- C07K16/082—Hepadnaviridae, e.g. hepatitis B virus
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Abstract
The invention discloses a kind of processing method of 4 resin columns of pharmaceutical grade XAD for HBsAg purifying, comprise the following steps：(1) 4 resins of XAD are cleaned with water for injection after removing suspended particulate and loads column casing；(2) when water for injection flushing 0.5 1.5 is small；(3) when the ethanol solution flushing 13 that volume fraction is 20% is small；(4) when water for injection flushing 0.5 1.5 is small；(5) when the HCl solution flushing 13 that molar concentration is 0.1mol/L is small；(6) when water for injection flushing 0.5 1.5 is small；(7) when the NaOH solution flushing 13 that molar concentration is 0.1mol/L is small；(8) more than when the Na0H solution preservation 6 that molar concentration is 0.1mol/L is small；(9) when small with the phosphate buffer solution flushing 35 of 6.2mmol/L before.4 resin columns of XAD after the method for the present invention processing can reach the requirement of pharmaceutical grade, have very strong triton adsorption capacity, and protein adsorption rate is small, and its cost is extremely cheap for 4 resin columns of pharmaceutical grade finished product XAD, has preferable economic benefit.
Description
Background technology
The present invention relates to a kind of processing method of the pharmaceutical grade XAD-4 resin columns for HBsAg purifying, belong to bio-pharmaceuticals
Technology.
Background technology
XAD-4 resins are a kind of nonpolar macroporous polystyrene adsorption resins, are usually used in from process water and polar solvent
Middle removing small organic molecule., it is necessary to change molecular surface tension force using surfactant-triton in HBsAg purifying process
To separate different components, XAD-4 resins are utilized to achieve the purpose that to remove triton to the Specific adsorption of triton in subsequent technique.
Available for purifying HBsAg pharmaceutical grade finished product XAD-4 resin columns, commercially available pharmaceutical grade finished product XAD-4 resin columns are currently mainly used,
Not only price is high, and is difficult to regeneration recycling, and production cost is very high, is unfavorable for the large-scale production of HBsAg.
The content of the invention
It is an object of the invention in order to solve shortcoming existing in the prior art, and provide a kind of for HBsAg purifying
The processing method of pharmaceutical grade XAD-4 resin columns.The present invention use technical grade XAD-4 resins in bulk as raw material, by ad hoc approach with
After flow processing, the requirement of pharmaceutical grade XAD-4 resin columns is can reach, there is very strong triton adsorption capacity, and protein adsorption rate
It is small, and its cost is extremely cheap for pharmaceutical grade finished product XAD-4 resin columns, has preferable economic benefit.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying, comprises the following steps：
(1) XAD-4 resins are cleaned with water for injection after removing suspended particulate and loads column casing；
(2) when water for injection flushing 0.5-1.5 is small；
(3) when the ethanol solution flushing 1-3 that volume fraction is 20% is small；
(4) when water for injection flushing 0.5-1.5 is small；
(5) when the HCl solution flushing 1-3 that molar concentration is 0.1mol/L is small；
(6) when water for injection flushing 0.5-1.5 is small；
(7) when the NaOH solution flushing 1-3 that molar concentration is 0.1mol/L is small；
(8) more than when the NaOH solution preservation 6 that molar concentration is 0.1mol/L is small；
(9) when small with the phosphate buffer solution flushing 3-5 of 6.2mmol/L before.
Column casing is disposed vertically when the processing method is rinsed, and flushing liquor bottom in and top out, flow control is in 250-300ml/
min；If should connect when handling more XAD-4 resin columns at the same time, can at most connect 5 XAD-4 resin columns.
The XAD-4 resins are technical grade XAD-4 resins in bulk.
Compared with prior art, the present invention has following beneficial effect：
XAD-4 resin columns after the method for the present invention processing can reach the requirement of pharmaceutical grade, after being handled using the method for the present invention
XAD-4 resin columns be used for when purifying HBsAg, there is very strong triton adsorption capacity, and protein adsorption rate is small, in removal triton
Ability and protein adsorption rate reach or even be slightly better than presently commercially available pharmaceutical grade finished product XAD-4 resin columns, and it is prepared into
This is far below the price of commercially available pharmaceutical grade finished product XAD-4 resin columns, has preferable economic benefit.
Specific embodiment mode
The following example is further intended to the embodiment that citing more specifically describes the present invention, it should be understood that institute
Embodiment is stated only for the explanation present invention, rather than is limited the scope of the invention in any way.
Embodiment
First, column and cleaning are filled
1. material：
XAD-4 resin column casings：Developed by our company and designed, material is stainless steel, and disassembly and assembly is quick and convenient, column casing
Capacity 1L.
XAD-4 resins：Commercially available technical grade XAD-4 resins in bulk
2. preparation method
Commercially available technical grade XAD-4 resins in bulk are cleaned with water for injection after removing suspended particulate and load column casing, then successively
Cleaned using following reagent：
(1) when water for injection flushing 1 is small；
When (2) 20% ethanol solutions flushing 2 is small；
(3) when water for injection flushing 1 is small；
(4) when the HCl solution flushing 2 of 0.1mol/L is small；
(5) when water for injection flushing 1 is small；
(6) when the NaOH solution flushing 2 of 0.1mol/L is small；
(7) more than when the NaOH solution preservation 6 of 0.1mol/L is small；
(8) when small with the phosphate buffer solution flushing 4 of 6.2mmol/L before.
Column casing is disposed vertically during flushing, flushing liquor bottom in and top out, flow 300ml/min；If more XAD-4 are handled at the same time
It should connect during resin column, to ensure that cleaning performance can at most connect 5 XAD-4 resin columns.
2nd, cleaning performance
The XAD-4 for make commercially available technical grade XAD-4 resins extract in bulk by following operation, preparing by the method for the present invention
Resin column extract, commercially available pharmaceutical grade finished product XAD-4 resin column extracts, detect the DVB of these three extracts
(Divinlybenzene, divinylbenzene) residual volume and TCO (chromatography chemicals).
Commercially available technical grade XAD-4 resins extract production method in bulk：Take in 2 grams of commercially available XAD-4 resins in bulk and add 6
When 95% ethanol of milliliter immersion 16 is small.Analyze the DVB residual volumes and TCO (chromatography chemicals) in leachate.
XAD-4 resin column extract production methods prepared by the method for the present invention：Take by the XAD-4 trees after above-mentioned technical finesse
One, fat column, therefrom take added in 2 grams of resins 6 milliliter of 95% ethanol immersion 16 it is small when.Analyze leachate in DVB residual quantities and
TCO。
Commercially available pharmaceutical grade finished product XAD-4 resin column extract production methods：In commercially available pharmaceutical grade XAD-4 resin columns
In, take added in 2 grams of resins 6 milliliter of 95% ethanol immersion 16 it is small when.Analyze the DVB residual volumes and TCO in leachate.
The DVB residual volumes for detecting above extract (detect the big pi bond in DVB molecules to detect DVB with UV scanning method
Residual volume) and TCO obtain as a result, the DVB residual volumes of technical grade XAD-4 resins in bulk apparently higher than other two kinds leaching
Thing, and contain a large amount of other impurities, the DVB residual volumes of the XAD-4 resin columns prepared by the method for the present invention and TCO (chromatography
Chemicals) it is basically identical with commercially available XAD-4 resin columns.
In addition, phosphate buffer solution flushing 4 of the XAD-4 resin columns of the method for the present invention preparation in 6.2mmol/L is small i.e.
Sampling detection pH value, bacterial endotoxin, the pH value of its flushing liquor are consistent with the phosphate buffer solution of 6.2mmol/L before terminating
Or very close to the small 5EU/ml of bacterial endotoxin, meets HBsAg production technology needs.
Test example 1：XAD-4 resin columns after the method for the present invention processing are applied in purifying HBsAg techniques
In the purifying process of two batches, it would be desirable to which the product for removing triton is divided into two parts, and half is used by this
XAD-4 resin columns prepared by inventive method remove triton (experiment batch), the other half by normal process with commercially available pharmaceutical grade into
Product XAD-4 resin columns remove triton (control batch), and two parts product independently carries out follow-up production technology after removing triton
Until purifying final products, the effect of the XAD-4 resin columns prepared according to verification result to the method for the present invention are assessed.Following
During ring removes triton, it is sampled once per hour, detects the triton content in product, investigates XAD- prepared by the method for the present invention
4 resin columns and commercially available pharmaceutical grade finished product XAD-4 resin columns go triton ability, when circulation 4 is small after with the phosphorus of appropriate 6.2mmol/L
Hydrochlorate buffer solution cleans XAD-4 resin columns, and the front and rear product protein concentration of sampling detection XAD-4 circulations, investigates present invention side
XAD-4 resin columns prepared by method to the adsorption rate of albumen and purify final products with commercially available pharmaceutical grade finished product XAD-4 resin columns
Protein yield.Its testing result is as follows.
Table one：Triton content (unit in XAD-4 circulation different time products：μg/ml)
| Control batch 1 | Experiment batch 1 | Control batch 2 | Experiment batch 2 | |
| Before XAD-4 circulations | 1178 | 1182 | 1209 | 1209 |
| When circulating 1 hour | 700 | 599 | 846 | 698 |
| When circulating 2 hours | 474 | 387 | 574 | 430 |
| When circulating 3 hours | 361 | 329 | 473 | 356 |
| When circulating 4 hours | 329 | 291 | 404 | 340 |
| Total triton removal rate (%) | 72.1% | 75.4% | 66.6% | 71.9% |
The as shown by data of upper table：The ability that XAD-4 resin columns prepared by the method for the present invention remove triton is more medicinal than commercially available
Level XAD-4 resin columns be eager to excel, circulation 4 it is small when after, the average removal rate of triton in product：XAD-4 trees prepared by this technology
Fat column is 73.7%, and commercially available pharmaceutical grade finished product XAD-4 resin columns are 69.4%, therefore the XAD- prepared using the method for the present invention
Triton removal rate of 4 resin columns when XAD-4 is circulated is slightly above commercially available pharmaceutical grade finished product XAD-4 resin columns, contributes to product matter
The raising of amount.
Table two：The protein yield of the protein content of product and purifying final products (SFP) before and after XAD-4 circulations
As can be known from the above table：The average protein yield of XAD-4 resin columns prepared by the method for the present invention is 96.2%, commercially available medicinal
Level finished product XAD-4 resin column average proteins yield is 96.0%, and the protein content that the method for the present invention prepares the absorption of XAD-4 resin columns omits
Few, i.e., loss of proteins caused by when XAD-4 is circulated is smaller.XAD-4 circulation before product A to purify final products yield
(gram/KG), the experiment criticize flat average that the XAD-4 resin columns prepared with this technology produce is：(0.170+0.203) ÷ 2=
0.187, and the average value normally criticized is：(0.165+0.204) ÷ 2=0.185, i.e. experiment batch are higher by 1.1% than normally criticizing, without aobvious
Write difference, also illustrate that production in using the method for the present invention prepare XAD-4 resin columns for purify final products yield with
Commercially available pharmaceutical grade finished product XAD-4 resin columns are relatively basically identical.
Compareed by two batches technique, in triton removal rate, the adsorption rate of XAD-4 resin column albumen and final purified product
In terms of protein yield, XAD-4 resin columns prepared by the method for the present invention reach or even are slightly better than commercially available pharmaceutical grade finished product XAD-4 trees
Fat column.In addition, the purifying final products verification result of two experiments batch is qualified, show the XAD-4 resins prepared using this technology
Column will not have an impact the quality for purifying final products.
Experimental example 2：The quality of two kinds of XAD-4 resins tree purifying final products (SPF) is looked back
XAD-4 resin columns remove triton ability and directly affect the triton residual quantity purified in final products, may indirect shadow
SPF is rung than living, and than work=HBsAg concentration/total protein concentration, looking back XAD-4 resin columns input prepared by the method for the present invention makes
With preceding, lived with the ratio of commercially available pharmaceutical grade XAD-4 resin columns continuous 10 crowdes of SFP recently and the triton residual quantity of SFP and side of the present invention
Method prepare XAD-4 resin columns come into operation after initial 10 crowdes of SPF ratio live and SFP triton residual quantity, comparative analysis two
Influence of the kind XAD-4 resin columns to final purified product SPF.
Table three：Commercially available XAD-4 resin columns go the ratio of continuous 10 final products of triton production to live and triton residual quantity
Table four：XAD-4 resin columns prepared by the method for the present invention go the ratio of continuous 10 final products of triton production living and bent
Logical residual quantity
Show in the analysis result of engineer testing and retrospective verification, the XAD-4 resin columns prepared with the method for the present invention are gone
It is 0.744 that the purifying final products average specific that triton is produced, which is lived, and the final production of purifying is produced with commercially available XAD-4 resin columns
The work 0.739 of product average specific is basically identical, in the range of than criterion of acceptability (>=0.5) living；Prepared with the method for the present invention
The purifying final products that XAD-4 resin columns go triton the to produce triton content that is averaged is 11.84 μ g/ml, also with commercially available XAD-4
It is basically identical that resin column produces the purifying final products 11.76 μ g/ml of triton content that be averaged, far low criterion of acceptability (≤25 μ
G/ml), it is seen that up to commercially available pharmaceutical grade sold resin when XAD-4 resin columns prepared by the method for the present invention are used to purify HBsAg
Equivalent effect, will not produce harmful effect to product quality.
The using effect of XAD-4 resin columns prepared by the method for the present invention can reach commercially available pharmaceutical grade finished product XAD-4 resins
The using effect of column, therefore the available commercially available pharmaceutical grade finished product XAD-4 resin columns of replacement are used to produce recombinant hepatitis B vaccine
(saccharomyces cerevisiae).Its manufacturing cost is prepared medicinal in the process of the present invention well below commercially available pharmaceutical grade finished product XAD-4 resin columns
Level XAD-4 resin columns have significant economic benefit for purifying HBsAg.
Claims (3)
1. the processing method of a kind of pharmaceutical grade XAD-4 resin columns for HBsAg purifying, it is characterised in that comprise the following steps：
(1) XAD-4 resins are cleaned with water for injection after removing suspended particulate and loads column casing；
(2) when water for injection flushing 0.5-1.5 is small；
(3) when the ethanol solution flushing 1-3 that volume fraction is 20% is small；
(4) when water for injection flushing 0.5-1.5 is small；
(5) when the HCl solution flushing 1-3 that molar concentration is 0.1mol/L is small；
(6) when water for injection flushing 0.5-1.5 is small；
(7) when the Na0H solution flushing 1-3 that molar concentration is 0.1mol/L is small；
(8) more than when the NaOH solution preservation 6 that molar concentration is 0.1mol/L is small；
(9) when small with the phosphate buffer solution flushing 3-5 of 6.2mmol/L before.
2. a kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying according to claim 1, it is special
Sign is that column casing is disposed vertically when the processing method is rinsed, and flushing liquor bottom in and top out, flow control is in 250-300ml/min；
If should connect when handling more XAD-4 resin columns at the same time, can at most connect 5 XAD-4 resin columns.
3. a kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying according to claim 1, it is special
Sign is that the XAD-4 resins are technical grade XAD-4 resins in bulk.
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| CN201711212402.8A CN107970899A (en) | 2017-11-27 | 2017-11-27 | A kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying |
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| CN201711212402.8A CN107970899A (en) | 2017-11-27 | 2017-11-27 | A kind of processing method of pharmaceutical grade XAD-4 resin columns for HBsAg purifying |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01149795A (en) * | 1987-12-04 | 1989-06-12 | Toyo Seito Kk | Method for purifying alpha-glycosylglycyrrhizin |
| CN101723805A (en) * | 2009-12-08 | 2010-06-09 | 浙江工业大学 | Separation method for preparing natural 2-phenylethyl alcohol by biotransformation method |
| CN101979530A (en) * | 2010-09-26 | 2011-02-23 | 湖北国力生物技术开发有限公司 | Method suitable for industrialized production for separating and purifying natto kinase |
| CN103333938A (en) * | 2013-07-19 | 2013-10-02 | 深圳康泰生物制品股份有限公司 | Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine |
| CN103805645A (en) * | 2014-03-09 | 2014-05-21 | 桂林理工大学 | Separation and purification preparation method of water-soluble antibiotic |
-
2017
- 2017-11-27 CN CN201711212402.8A patent/CN107970899A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01149795A (en) * | 1987-12-04 | 1989-06-12 | Toyo Seito Kk | Method for purifying alpha-glycosylglycyrrhizin |
| CN101723805A (en) * | 2009-12-08 | 2010-06-09 | 浙江工业大学 | Separation method for preparing natural 2-phenylethyl alcohol by biotransformation method |
| CN101979530A (en) * | 2010-09-26 | 2011-02-23 | 湖北国力生物技术开发有限公司 | Method suitable for industrialized production for separating and purifying natto kinase |
| CN103333938A (en) * | 2013-07-19 | 2013-10-02 | 深圳康泰生物制品股份有限公司 | Recombinant saccharomyces-fermentum-expressed hepatitis B surface antigen, production method of hepatitis B surface antigen, hepatitis B vaccine and production method of hepatitis B vaccine |
| CN103805645A (en) * | 2014-03-09 | 2014-05-21 | 桂林理工大学 | Separation and purification preparation method of water-soluble antibiotic |
Non-Patent Citations (3)
| Title |
|---|
| 刘香萍: "《紫丁香主要活性成分制备及抗氧化应激活性评价》", 30 September 2015, 中国农业大学出版社 * |
| 张文清: "《分离分析化学》", 28 February 2007, 华东理工大学出版社 * |
| 蔡宝昌等: "《中药制剂前处理新技术与新设备》", 30 November 2005, 中国医药科技出版社 * |
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