CN105126131A - Sterilization method of protein A immuno-absorbent column - Google Patents
Sterilization method of protein A immuno-absorbent column Download PDFInfo
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- CN105126131A CN105126131A CN201510556533.2A CN201510556533A CN105126131A CN 105126131 A CN105126131 A CN 105126131A CN 201510556533 A CN201510556533 A CN 201510556533A CN 105126131 A CN105126131 A CN 105126131A
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Abstract
The invention relates to a sterilization method of a protein A immuno-absorbent column. The sterilization method comprises the following steps: adding a protein A immuno-absorbent to a buffer solution which is 4-7 in pH value, packing a column and sterilizing with steam. The method further comprises a step of adding a stabilizer to the buffer solution, wherein the stabilizer consists of the following components: an antioxidant, polyhydric alcohol, polysaccharide, a chelating agent, a thickening agent, polypeptide or protein. By virtue of the sterilization process, the activity of the absorbent can be protected after sterilization, and the final sterilization of the protein A immuno-absorbent column can be achieved; and the obtained protein A immuno-absorbent column product, compared with products obtained from a sterile process, is higher in sterility assurance level and is lower in production cost.
Description
Technical field
The present invention relates to biomaterial and field of blood purification, particularly relate to the sterilizing methods of Protein A immunoadsorption post.
Background technology
Protein A immunoadsorption post is mainly used in treating autoimmune disease.Autoimmune disease refers to that the immune system of human body gets muddled, by the material of self as exotic antigen, create a series of immunoreation, cause the generation of a large amount of autoantibody and immune complex in body, and then destroy the general name of a large class disease of tissue, organ.Comprise myasthenia gravis, systemic lupus erythematosus (sle), rheumatoid arthritis, Guillain Barre syndrome etc. more than 80 and plant disease.Because the cause of disease is unclear, at present effective drug treatment is still lacked to such disease.Utilize the autoantibody in the direct adsorption removal patient blood of Protein A immunoadsorption post and immune complex, be acknowledged as the most effectual way of such disease for the treatment of at present.Protein A is the cell wall protein of staphylococcus aureus, the Fc section of this albumen and antibody has specificity to interact, make use of the high selectivity effect of protein A antagonist just, the affinity adsorption column utilizing immobilization protein A to synthesize can realize the quick removing to immune complex most of in autoimmune disease patient blood and autoantibody.In the clinical practice of more than ten years, Protein A immunoadsorption post has demonstrated the incomparable unique curative effect of Drug therapy in treatment autoimmune disease.
Protein A immunoadsorption post belongs to three class medical apparatus and instruments, must ensure the aseptic of product.Protein A immunoadsorption post on market mainly contains Immunosorb and Prosorb two kinds that Fresenius company produces, because the adsorbent in Protein A immunoadsorption post has sterilizing sensitivity, therefore generally Protein A immunoadsorption post can not carry out pressure sterilizing or irradiation sterilization, and its production process adopts aseptic processing.The recombinant protein A of special acid sequence can be obtained by the method for genetic modification, utilize the adsorbent of the white A synthesis of this egg size group can tolerate steam sterilization (CN102398717A).
Its technological process of aseptic processing is complicated, sterility assurance level lower (sterility assurance level of aseptic processing is one thousandth, and the sterility assurance level of terminal sterilization is 1,000,000/), once microbiological contamination is also difficult to save loss.And the acquisition of tolerance type recombinant protein A needs to carry out genetic engineering modified, go for desirable destination protein workload comparatively large, the stability of destination protein also needs to take into full account.
For perfusion device product, best sterilizing methods is steam sterilization and irradiation sterilization, considerable perfusion device product all adopts this two kinds of methods in the market, but for Protein A immunoadsorption post, process because of this extreme condition can cause the degraded degeneration of active component protein A in adsorption column, thus loses the ability removing morbid substance from blood.
Therefore, for a kind of feasible sterilization process of Protein A immunoadsorption post searching is necessary.
Summary of the invention
Based on this, the object of the present invention is to provide a kind of sterilizing methods of Protein A immunoadsorption post, to realize the sterilizing of Protein A immunoadsorption post.
The object of the invention is achieved through the following technical solutions:
A sterilizing methods for Protein A immunoadsorption post, is characterized in that, comprises the following steps: Protein A immunoadsorption agent is placed in the buffer that pH is 4 ~ 7, carries out steam sterilization after dress post.
Wherein in some embodiments, described method is also included in described buffer adds stabilizing agent, and described stabilizing agent is composed of the following components: antioxidant, polyhydric alcohol, polysaccharide, chelating agen, thickening agent, polypeptide or albumen.
Wherein in some embodiments, described stabilizing agent is made up of the component of following weight portion: sodium ascorbate 0.2 ~ 5 part or sodium pyrosulfite 0.05 ~ 2 part, glycerol 2 ~ 40 parts, stevioside 1 ~ 20 part, ethylenediaminetetraacetic acid 0.01 ~ 2 part, gelatin hydrolysate 0.1 ~ 10 part, albumin 0.1 ~ 2 part; The concentration of described stabilizing agent in buffer is 0.0326 ~ 0.79g/mL.
Wherein in some embodiments, described stabilizing agent is made up of the component of following weight portion: sodium pyrosulfite 0.1 ~ 0.3 part, glycerol 15 ~ 25 parts, stevioside 13 ~ 15 parts, ethylenediaminetetraacetic acid 0.05 ~ 0.2 part, gelatin hydrolysate 1 ~ 3 part, albumin 0.1 ~ 0.3 part; The concentration of described stabilizing agent in buffer is 0.2925 ~ 0.438g/mL.
Wherein in some embodiments, described stabilizing agent is made up of the component of following weight portion: sodium pyrosulfite 0.2 part, glycerol 20 parts, stevioside 15 parts, gelatin hydrolysate 2 parts, bovine serum albumin 0.2 part, ethylenediaminetetraacetic acid 0.1 part; The concentration of described stabilizing agent in buffer is 0.375g/mL.
Wherein in some embodiments, described buffer is selected from citrate buffer solution, acetate buffer, phosphate buffer, glycine buffer, tartaric acid buffer, maleate buffer, borate buffer, TRIS buffer or ethanesulfonic acid buffer.
Wherein in some embodiments, the ion concentration of described buffer is 0.1 ~ 0.5M.
Wherein in some embodiments, the pH of described buffer is 5 ~ 6.
The steam sterilization condition used in the present invention is 121 DEG C, 15min (being equivalent to normal sterilization time F0=15min), the steam sterilization condition of the reduction used in the present invention is 115 DEG C, 30min (being equivalent to normal sterilization time F0=8min) and 100 DEG C, 60min (flowing steam sterilization condition, can as assisted sterilization means).
The pH value used in the present invention is 4 ~ 7, and preferable ph is 5 ~ 6.PH plays a role protein A conformational stability for the pH scope that the activity of adsorbent after maintenance sterilizing is extremely important, suitable.
The ion concentration of the buffer used in the present invention is 0.02 ~ 2.0M, preferably 0.1 ~ 0.5M, and this concentration is useful for the activity of adsorbent after maintenance sterilizing, and the bin stability for adsorbent is also useful.Concentration is too low, and buffer capacity is not enough; , may there is precipitation in excessive concentration, in addition, the flushing before also giving Clinical practice is made troubles in storage process.
The stabilizing agent used in the present invention has antioxidant, polyhydric alcohol, polysaccharide, chelating agen, albumin, thickening agent.Antioxidant can prevent bioactive ingredients protein A in adsorbent oxidized under sterilising conditions; Polyhydric alcohol and polysaccharide can be explained with " preferential exclusion effect " as the mechanism of stabilizing agent, namely when polyhydric alcohol or polysaccharide exist, its polyhydroxy and solvent molecule form hydrogen bond, the surface tension of solvent is increased, solution viscosity increases, and produces exclusion effect to protein, and the hydration shell of albumen reduces, structure is compacter, thus increases the stability of albumen; Chelating agen can in complex solution some to the influential metal ion of protein active; Albumin belongs to protide protective agent, is of wide application as classical, excellent stabilizing agent; Aggregate and precipitate under the high temperature conditions when thickening agent obviously can prevent albumin individualism.
Therefore, the present invention has following beneficial effect:
Sterilizing methods of the present invention; the degraded degeneration of the active component protein A in sterilization process in protein A adsorption column can be prevented; maintain the stability of protein A; by this sterilization process; the activity of adsorbent can be made to be protected after sterilization; thus solve Protein A immunoadsorption post can not the problem of sterilizing, the final sterilization of Protein A immunoadsorption post product can be realized.
The Protein A immunoadsorption post product that sterilization process of the present invention obtains, compared with the product that aseptic processing obtains, sterility assurance level is higher, and production cost is lower.
Accompanying drawing explanation
Fig. 1 is the adsorption activity figure of protein A adsorbent under condition of different pH after sterilizing of embodiment 1;
Fig. 2 be embodiment 2 protein A adsorbent sterilizing before and after adsorption activity figure;
Fig. 3 be embodiment 3 protein A adsorbent sterilizing before and after adsorption activity figure;
Fig. 4 be embodiment 4 protein A adsorbent sterilizing before and after adsorption activity figure;
Fig. 5 is the protein A adsorbent of embodiment 5 adsorption activity figure before and after sterilizing under different sterilising conditions.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
The sterilizing methods of the present embodiment is as follows:
Get 5g protein A adsorbent, adding 5ml ion concentration is 0.2M, (wherein citrate buffer solution is selected in pH3 ~ 6 to the buffer of pH3 ~ 10, phosphate buffer is selected in pH7 ~ 8, Glycine-NaOH buffer is selected in pH9 ~ 10), bottling steam sterilization, sterilising conditions is 100 DEG C, 60min.
Then the absorption property of adsorbent before and after sterilizing is detected as follows:
By clean for adsorbent massive laundering, get 4.4ml and fill post, the balance liquid of 10 times of column volumes (1 × PBS, pH7.4) is first used to balance pillar, use the eluent of 10 times of column volumes (glycine of 0.1M, pH2.5) to rinse pillar again, then balance pillar with the balance liquid of 10 times of column volumes.Get 17ml blood plasma and cross post, flow velocity 1.7ml/min, first wash away non-associated proteins with balance liquid after having crossed blood plasma, then use elution associated proteins, collect eluting peak, detect protein concentration and calculate the absorption property of adsorbent.
The results are shown in Figure 1, as can be seen from Figure 1,100 DEG C, under the sterilising conditions of 60min, in the scope that pH is 3 ~ 10, increase with pH value, the retention of adsorbent activity first increases and reduces, and wherein when pH is 5 ~ 6, adsorbent activity retention is maximum.
Embodiment 2
The sterilizing methods of the present embodiment is as follows:
Get 5g protein A adsorbent, add the citrate buffer solution (or normal saline) that 5ml ionic strength is 0.2M, pH6.0, bottling steam sterilization, sterilising conditions is 121 DEG C, 15min (being equivalent to normal sterilization time F0=15min).
Then the absorption property of adsorbent before and after sterilizing is detected by the method for embodiment 1, the results are shown in Figure 2, as can be seen from Figure 2,121 DEG C, under the sterilising conditions of 15min, compared with non-sterilizing group, make the protein A adsorbent sterilizing of medium of normal saline after, loss of activity very large (activity retention is only 28%), and after adding the protein A adsorbent sterilizing of buffer, can activity recovery (activity retention returns to 70%) largely.
Embodiment 3
The sterilizing methods of the present embodiment is as follows:
Get 5g protein A adsorbent, add the citrate buffer solution (or normal saline) that 5ml ionic strength is 0.2M, pH6.0, bottling steam sterilization, sterilising conditions is 115 DEG C, 30min (being equivalent to normal sterilization time F0=8min).
Then the absorption property of adsorbent before and after sterilizing is detected by the method for embodiment 1, the results are shown in Figure 3, as can be seen from Figure 3,115 DEG C, under the sterilising conditions of 30min, compared with non-sterilizing group, make the protein A adsorbent sterilizing of medium of normal saline after, activity retention is 39%, and after adding the protein A adsorbent sterilizing of buffer, activity retention is 75%.Contrast known with embodiment 2, the steam sterilization condition of reduction is conducive to the reservation of adsorbent activity.
Embodiment 4
The sterilizing methods of the present embodiment is as follows:
Get 5g protein A adsorbent, add the citrate buffer solution (or normal saline) that 5ml ionic strength is 0.2M, pH6.0, bottling steam sterilization, sterilising conditions is 100 DEG C, 60min (flowing steam sterilization condition, can as assisted sterilization means).
Then the absorption property of adsorbent before and after sterilizing is detected by the method for embodiment 1, the results are shown in Figure 4, as can be seen from Figure 4,100 DEG C, under the sterilising conditions of 60min, compared with non-sterilizing group, make the protein A adsorbent sterilizing of medium of normal saline after, activity retention is 52%, and after adding the protein A adsorbent sterilizing of buffer, activity retention is 81%.Contrast known with embodiment 2 and embodiment 3, the condition of steam sterilization is gentleer, and adsorbent after sterilization activity retention is higher.
Embodiment 5
The sterilizing methods of the present embodiment is as follows:
Get 5g protein A adsorbent, adding 5ml ionic strength is 0.2M, the citrate buffer solution of pH6.0, (concentration of stabilizing agent in buffer is 0.375g/mL to be added with stabilizing agent in buffer, the weight portion of stabilizing agent consists of: 0.2 part of sodium pyrosulfite, 20 parts of glycerol, 15 parts of steviosides, 2 parts of gelatin hydrolysates, 0.2 part of BSA (bovine serum albumin), 0.1 part of ethylenediaminetetraacetic acid), bottling steam sterilization, sterilising conditions is respectively 121 DEG C/15min, 115 DEG C/30min, 100 DEG C/60min.
Then the absorption property of adsorbent before and after sterilizing is detected by the method for embodiment 1, the results are shown in Figure 5, as can be seen from Figure 5, under above-mentioned sterilising conditions, compared with non-sterilizing group, make the protein A adsorbent sterilizing of medium of normal saline after, loss of activity very large (see embodiment 2,3,4), and after adding the protein A adsorbent sterilizing of buffer and stabilizing agent, largely can recover the activity of adsorbent simultaneously, sterilising conditions is gentleer, and activation recovering degree is larger.Contrast known with embodiment 2,3,4, under steam sterilization condition, in buffer, add the reservation that stabilizing agent is conducive to protein A adsorbent activity.
Each technical characteristic of the above embodiment can combine arbitrarily, for making description succinct, the all possible combination of each technical characteristic in above-described embodiment is not all described, but, as long as the combination of these technical characteristics does not exist contradiction, be all considered to be the scope that this description is recorded.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be construed as limiting the scope of the patent.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (8)
1. a sterilizing methods for Protein A immunoadsorption post, is characterized in that, comprises the following steps: Protein A immunoadsorption agent is placed in the buffer that pH is 4 ~ 7, carries out steam sterilization after dress post.
2. the sterilizing methods of Protein A immunoadsorption post according to claim 1, it is characterized in that, also be included in described buffer and add stabilizing agent, described stabilizing agent is composed of the following components: antioxidant, polyhydric alcohol, polysaccharide, chelating agen, thickening agent, polypeptide or albumen.
3. the sterilizing methods of Protein A immunoadsorption post according to claim 2, it is characterized in that, described stabilizing agent is made up of the component of following weight portion: sodium ascorbate 0.2 ~ 5 part or sodium pyrosulfite 0.05 ~ 2 part, glycerol 2 ~ 40 parts, stevioside 1 ~ 20 part, ethylenediaminetetraacetic acid 0.01 ~ 2 part, gelatin hydrolysate 0.1 ~ 10 part, albumin 0.1 ~ 2 part; The concentration of described stabilizing agent in buffer is 0.0326 ~ 0.79g/mL.
4. the sterilizing methods of Protein A immunoadsorption post according to claim 3, it is characterized in that, described stabilizing agent is made up of the component of following weight portion: sodium pyrosulfite 0.1 ~ 0.3 part, glycerol 15 ~ 25 parts, stevioside 13 ~ 15 parts, ethylenediaminetetraacetic acid 0.05 ~ 0.2 part, gelatin hydrolysate 1 ~ 3 part, albumin 0.1 ~ 0.3 part; The concentration of described stabilizing agent in buffer is 0.2925 ~ 0.438g/mL.
5. the sterilizing methods of Protein A immunoadsorption post according to claim 4, it is characterized in that, described stabilizing agent is made up of the component of following weight portion: sodium pyrosulfite 0.2 part, glycerol 20 parts, stevioside 15 parts, gelatin hydrolysate 2 parts, bovine serum albumin 0.2 part, ethylenediaminetetraacetic acid 0.1 part; The concentration of described stabilizing agent in buffer is 0.375g/mL.
6. the sterilizing methods of the Protein A immunoadsorption post according to any one of claim 1-5, it is characterized in that, described buffer is selected from citrate buffer solution, acetate buffer, phosphate buffer, glycine buffer, tartaric acid buffer, maleate buffer, borate buffer, TRIS buffer or ethanesulfonic acid buffer.
7. the sterilizing methods of the Protein A immunoadsorption post according to any one of claim 1-5, is characterized in that, the ion concentration of described buffer is 0.1 ~ 0.5M.
8. the sterilizing methods of the Protein A immunoadsorption post according to any one of claim 1-5, is characterized in that, the pH of described buffer is 5 ~ 6.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110665478A (en) * | 2019-09-06 | 2020-01-10 | 武汉瑞法医疗器械有限公司 | Filling liquid for stabilizing adsorption performance of adsorbent and application thereof |
| CN110711276A (en) * | 2019-09-06 | 2020-01-21 | 武汉瑞法医疗器械有限公司 | Preserving fluid for stabilizing adsorption performance of adsorbent |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102698717A (en) * | 2012-05-11 | 2012-10-03 | 广州康盛生物科技有限公司 | Protein A adsorption medium |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102698717A (en) * | 2012-05-11 | 2012-10-03 | 广州康盛生物科技有限公司 | Protein A adsorption medium |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110665478A (en) * | 2019-09-06 | 2020-01-10 | 武汉瑞法医疗器械有限公司 | Filling liquid for stabilizing adsorption performance of adsorbent and application thereof |
| CN110711276A (en) * | 2019-09-06 | 2020-01-21 | 武汉瑞法医疗器械有限公司 | Preserving fluid for stabilizing adsorption performance of adsorbent |
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