JP2537935B2 - Agricultural reduction and desalination method for physiologically active substances - Google Patents
Agricultural reduction and desalination method for physiologically active substancesInfo
- Publication number
- JP2537935B2 JP2537935B2 JP62334046A JP33404687A JP2537935B2 JP 2537935 B2 JP2537935 B2 JP 2537935B2 JP 62334046 A JP62334046 A JP 62334046A JP 33404687 A JP33404687 A JP 33404687A JP 2537935 B2 JP2537935 B2 JP 2537935B2
- Authority
- JP
- Japan
- Prior art keywords
- physiologically active
- collagen
- concentration
- desalting
- active protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims description 31
- 239000013543 active substance Substances 0.000 title description 26
- 238000010612 desalination reaction Methods 0.000 title description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 108010035532 Collagen Proteins 0.000 claims description 27
- 102000008186 Collagen Human genes 0.000 claims description 27
- 229920001436 collagen Polymers 0.000 claims description 27
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 238000011033 desalting Methods 0.000 claims description 18
- 238000000502 dialysis Methods 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- 108010045569 atelocollagen Proteins 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 102000007644 Colony-Stimulating Factors Human genes 0.000 claims description 2
- 108010071942 Colony-Stimulating Factors Proteins 0.000 claims description 2
- 102000004127 Cytokines Human genes 0.000 claims description 2
- 108090000695 Cytokines Proteins 0.000 claims description 2
- 108090000394 Erythropoietin Proteins 0.000 claims description 2
- 102000003951 Erythropoietin Human genes 0.000 claims description 2
- 108010051696 Growth Hormone Proteins 0.000 claims description 2
- 102000018997 Growth Hormone Human genes 0.000 claims description 2
- 102000038461 Growth Hormone-Releasing Hormone Human genes 0.000 claims description 2
- 239000000095 Growth Hormone-Releasing Hormone Substances 0.000 claims description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- 102000014150 Interferons Human genes 0.000 claims description 2
- 108010050904 Interferons Proteins 0.000 claims description 2
- 102000000588 Interleukin-2 Human genes 0.000 claims description 2
- 108010002350 Interleukin-2 Proteins 0.000 claims description 2
- 102000001938 Plasminogen Activators Human genes 0.000 claims description 2
- 108010001014 Plasminogen Activators Proteins 0.000 claims description 2
- 101710142969 Somatoliberin Proteins 0.000 claims description 2
- 102000013275 Somatomedins Human genes 0.000 claims description 2
- 230000000975 bioactive effect Effects 0.000 claims description 2
- 229940105423 erythropoietin Drugs 0.000 claims description 2
- 239000000122 growth hormone Substances 0.000 claims description 2
- 229940079322 interferon Drugs 0.000 claims description 2
- 229940127126 plasminogen activator Drugs 0.000 claims description 2
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 2
- 239000012460 protein solution Substances 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 14
- 239000012528 membrane Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 102000006992 Interferon-alpha Human genes 0.000 description 9
- 108010047761 Interferon-alpha Proteins 0.000 description 9
- 239000000126 substance Substances 0.000 description 9
- 238000011084 recovery Methods 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 230000001766 physiological effect Effects 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 229920002492 poly(sulfone) Polymers 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000012832 cell culture technique Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N dihydromaleimide Natural products O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 229960002317 succinimide Drugs 0.000 description 1
- -1 succinimide ester Chemical class 0.000 description 1
Landscapes
- Filtration Of Liquid (AREA)
- Peptides Or Proteins (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は、生理活性たんぱく質を含有する溶液の濃縮
・脱塩方法に関し、さらに詳しくは、精製された生理活
性たんぱく質含有水溶液を効率良く濃縮・脱塩する方法
に関するものである。TECHNICAL FIELD The present invention relates to a method for concentrating and desalting a solution containing a physiologically active protein, more specifically, a method for efficiently concentrating a purified aqueous solution containing a physiologically active protein. It relates to a method of desalting.
［従来技術および問題点］ たんぱく質、ペプチド、糖たんぱく質またはホルモン
等のいわゆる生理活性物質は、微量でヒトおよび動物の
生理に大きく影響を及ぼす物質であり、種々の生理機能
の研究および臨床治療への応用等、様々な分野での重要
性がしばしば指摘されている。これらの生理活性物質
は、生体組織からの抽出、精製によって得られるが、組
織中の含有量が極めて微量にすぎない上、物理的な作用
に比較的不安定であるために濃縮および脱塩等の処理工
程中に失活したり、あるいは吸着等により損失し、実質
的な収率は極めて低い。従って臨床治療面においてはい
うまでもなく、研究面での需要にも充分応え得ないとい
う問題点がある。近年の細胞培養技術あるいは遺伝子組
換え技術の進歩により、有用な生理活性物質が従来より
大量に生産されるようになったが、このような方法によ
って得られる生理活性物質も、微妙な条件の変化で失活
され易いという点では従来と変わりはなく、精製および
濃縮等の工程に伴って実質的な収率の大幅な低下を避け
がたいのが実状である。さらに、精製工程中に溶液中の
生理活性物質が使用に不適当な程度にまで希釈された
り、溶液中に不必要な物質、特に塩類が混入されて実用
に適さなくなる場合も多い。このような理由から、脱塩
・濃縮して最終的に得られる製品は、最新の技術を駆使
しても非常に高価なものとなり、臨床上および研究上の
要求を充分満たすまでに至っていない。[Prior Art and Problems] So-called physiologically active substances such as proteins, peptides, glycoproteins or hormones are substances that greatly affect the physiology of humans and animals in a trace amount, and are useful for research and clinical treatment of various physiological functions. The importance in various fields such as application is often pointed out. These physiologically active substances are obtained by extraction and purification from living tissues, but their content in tissues is extremely small and their concentration and desalting are relatively unstable due to their physical action. Is inactivated during the treatment step, or is lost due to adsorption or the like, and the substantial yield is extremely low. Therefore, there is a problem that it is not possible to sufficiently meet the demand for research as well as for clinical treatment. Due to recent advances in cell culture technology or gene recombination technology, useful physiologically active substances have been produced in larger amounts than before, and physiologically active substances obtained by such methods also have subtle changes in conditions. It is the same as the conventional one in that it is easily deactivated, and the fact is that it is unavoidable that a substantial decrease in the yield is accompanied by the steps such as purification and concentration. Further, in many cases, the physiologically active substance in the solution is diluted to a degree unsuitable for use during the purification step, or unnecessary substances, especially salts are mixed in the solution, which makes it unsuitable for practical use. For these reasons, the final product obtained by desalting and concentrating becomes extremely expensive even by making full use of the latest technology, and has not yet fully met the clinical and research requirements.
通常、生理活性物質の濃縮および脱塩は凍結乾燥法、
沈殿法、イオン交換法、減圧透析法および限外濾過法党
を用いて行なわれている。しかしながら、凍結乾燥法は
脱塩に有効でなく、生理活性物質のみならず塩類および
低分子物質も同様に濃縮されるので、溶液中の塩濃度が
上昇し、生理活性物質の失活、変性も招く恐れが高い。
沈殿法は、大量の処理には適するが、収率が低い上、沈
殿剤の除去が問題である。イオン交換法は、大量の処理
に適し、また比較的容易に完全な脱塩を期待し得るが、
吸着による生理活性物質損失の恐れがある上、非イオン
性の低分子物質が除去されず、さらに他の緩衝液への転
用が困難であるという問題がある。減圧透析法は、少量
の試料の処理には適するが、大量の処理には不適当であ
る。また、限外濾過法は、大量処理に適し、装置も比較
的安価に得られるが、溶液中に微量しか存在しない生理
活性物質が吸着や剪断応力によって損失または失活され
る結果、収率が低下するという問題を有する。Usually, concentration and desalting of physiologically active substances are lyophilized,
It is carried out using precipitation methods, ion exchange methods, vacuum dialysis methods and ultrafiltration methods. However, the freeze-drying method is not effective for desalting, and not only physiologically active substances but also salts and low-molecular substances are similarly concentrated. Therefore, the salt concentration in the solution is increased, and the physiologically active substances are inactivated and denatured. There is a high risk of inviting.
Although the precipitation method is suitable for large-scale processing, the yield is low and the removal of the precipitating agent is a problem. The ion exchange method is suitable for large-scale processing, and although complete desalination can be expected relatively easily,
There is a problem that a physiologically active substance may be lost due to adsorption, a nonionic low molecular weight substance is not removed, and it is difficult to convert the substance to another buffer solution. The vacuum dialysis method is suitable for processing a small amount of sample, but is not suitable for processing a large amount of sample. In addition, the ultrafiltration method is suitable for large-scale processing, and the apparatus can be obtained relatively inexpensively, but as a result of loss or deactivation of physiologically active substances present in a solution in a trace amount due to adsorption or shear stress, the yield is increased. It has the problem of lowering.
このように、現行の濃縮・脱塩方法はいずれも生理活
性物質を含有する溶液の処理には不都合な点を有してお
り、従って、生理活性物質含有水溶液、特に精製後の生
理活性物質含有水溶液を、活性物質の失活や損失を伴な
わずに適当な濃度に濃縮および／または脱塩する方法の
開発が強く望まれていた。As described above, all of the current concentration / desalting methods have disadvantages in treating a solution containing a physiologically active substance, and therefore, an aqueous solution containing a physiologically active substance, especially a physiologically active substance-containing solution after purification, is present. It has been strongly desired to develop a method for concentrating and / or desalting an aqueous solution to an appropriate concentration without deactivating or losing the active substance.
［問題点を解決するための手段］ 本発明者らは、精製処理後の生理活性物質含有水溶液
を、該生理活性物質の失活および／または損失を伴うこ
となく高収率で、容易に濃縮・脱塩する方法を得ること
を目的として鋭意研究を重ねた結果、精製された生理活
性物質含有水溶液にコラーゲンを加え、この共存下で透
析または限外濾過すると、生理活性物質の失活や損失を
伴うことなく、効率良く濃縮・脱塩することができるこ
とを見い出し、本発明を完成するに至った。[Means for Solving Problems] The inventors of the present invention can easily concentrate a purified aqueous solution containing a physiologically active substance in a high yield without deactivation and / or loss of the physiologically active substance. -As a result of intensive studies aimed at obtaining a method for desalting, when collagen is added to a purified aqueous solution containing a physiologically active substance and dialysis or ultrafiltration is performed in the coexistence with this, inactivation or loss of the physiologically active substance is caused. It was found that the concentration and desalting can be efficiently carried out without causing the above, and the present invention has been completed.
本発明に於ける生理活性物質には、生理活性を有する
ポリペプチド、たんぱく質、糖たんぱく質などが含まれ
るが、本明細書では便宜上、これらを総括して「生理活
性たんぱく質」と呼ぶ。The physiologically active substance in the present invention includes polypeptides, proteins, glycoproteins and the like having physiological activity, but in the present specification, these are collectively referred to as "physiologically active protein" for convenience.
即ち、本発明は、精製された生理活性たんぱく質を含
有する水溶液に、コラーゲンを加え、得られた混合物を
限外濾過または透析することを特徴とする生理活性たん
ぱく質水溶液の濃縮・脱塩方法を提供するものである。That is, the present invention provides a method for concentrating and desalting an aqueous physiologically active protein solution, which comprises adding collagen to an aqueous solution containing a purified biologically active protein and subjecting the resulting mixture to ultrafiltration or dialysis. To do.
本発明方法に用いられるコラーゲンは、生理活性たん
ぱく質含有水溶液中の塩類および低分子物質のみが、透
析膜または限外濾過膜を介して効率良く排除されるよう
に作用し、もって該物質の損失を防ぐ。このコラーゲン
は、生理活性たんぱく質と共に溶液中に残存する。しか
し、コラーゲンはヒトまたは動物にとって無毒であると
共に、自身に生理活性が殆んどまたは全く無い物質、も
しくは生理活性物質との分離が容易である物質である。The collagen used in the method of the present invention acts so that only salts and low-molecular substances in the physiologically active protein-containing aqueous solution are efficiently eliminated through the dialysis membrane or the ultrafiltration membrane, and thus the loss of the substances is caused. prevent. This collagen remains in solution along with the bioactive protein. However, collagen is a substance that is non-toxic to humans or animals, has little or no physiological activity, or is easily separated from a physiologically active substance.
上記の条件に適合する限り、本発明方法に用い得るコ
ラーゲン、ゼラチンまたはそれらの混合物のうち、特に
好ましいのはコラーゲンである。コラーゲンは、pH調整
や塩濃度調整で容易に生理活性物質との分離が可能であ
る。コラーゲンは、それらが元来抗原性の低い物質であ
る上、現在入手可能な注射用コラーゲンは、ウシ真皮か
ら抽出したコラーゲンをペプシン処理して抗原決定基と
なるテロペプチドを除去したものであることから、本発
明方法に用いさらにはヒトに投与することにおいてなん
ら問題の無い物質といえる。従って、コラーゲンは、い
かなる種に由来するものであってもよい。なお、コラー
ゲンは、分子中のリジン残基のε−アミノ酸をカルボキ
シル基に変換したり、カルボキシル基をサクシンイミド
エステルに変換して活性化した誘導体、あるいは、遺伝
子組換え技術で生産されたものであってもよい。Of the collagen, gelatin or mixtures thereof that can be used in the method of the present invention, collagen is particularly preferred as long as it meets the above conditions. Collagen can be easily separated from physiologically active substances by adjusting pH and salt concentration. Collagen is originally a substance with low antigenicity, and currently available collagen for injection is obtained by treating collagen extracted from bovine dermis with pepsin to remove the telopeptide that serves as an antigenic determinant. Therefore, it can be said that there is no problem in using it in the method of the present invention and further administering it to humans. Therefore, the collagen may be from any species. Collagen is a derivative obtained by converting the ε-amino acid of a lysine residue in the molecule into a carboxyl group, or converting the carboxyl group into a succinimide ester and activated, or produced by gene recombination technology. It may be.
生理活性たんぱく質に対するコラーゲンの割合は、厳
密なものではないが、通常、生理活性たんぱく質１モル
に対し、約10-4モル以上の割合で加えるとよい。具体的
な添加量は、処理すべき溶液の生理活性たんぱく質の種
類および濃度、並びに選択されたコラーゲン等に左右さ
れる。一般にコラーゲンの場合、めやすとしては、生理
活性たん白質に対し、モル比で５×10-4〜２×10-4程度
である。また添加濃度は約0.0001〜２％（w/v）である
ことが好ましく、さらに、0.001〜１％（w/v）であるこ
とがより好ましい。また、添加濃度は約0.001〜20（w/
v）％であることが好ましく、さらに、0.01〜５％（w/
v）であることがより好ましい。The ratio of collagen to the physiologically active protein is not critical, but it is usually preferable to add collagen at a ratio of about 10 −4 mol or more to 1 mol of the physiologically active protein. The specific amount to be added depends on the type and concentration of the physiologically active protein in the solution to be treated, the selected collagen and the like. Generally, in the case of collagen, the aim is about 5 × 10 −4 to 2 × 10 −4 in molar ratio with respect to the physiologically active protein. Further, the added concentration is preferably about 0.0001 to 2% (w / v), and more preferably 0.001 to 1% (w / v). The concentration of addition is about 0.001 to 20 (w /
v)% is preferable, and 0.01 to 5% (w /
v) is more preferable.
本発明方法は特定の生理活性たんぱく質を含有する溶
液の濃縮・脱塩処理に限定されないが、微量で生理活性
を有し、物理的な作用に比較的不安定なたんぱく質の濃
縮・脱塩において特に有用である。それらの活性たんぱ
く質は、通常の濃縮・脱塩法では失活したり、吸着によ
って損失され、充分な回収率を期待することができな
い。この様なたんぱく質の代表的な例として、インター
フェロン、コロニー形成刺激因子、成長ホルモン、成長
ホルモン放出因子、インシュリン様成長因子、プラスミ
ノーゲン活性化因子、エリスロポエチン、インターロイ
キン２等のサイトカインを挙げることができる。The method of the present invention is not limited to the concentration / desalting treatment of a solution containing a specific physiologically active protein, but particularly in the concentration / desalting of a protein having a small amount of physiological activity and relatively unstable to physical action. It is useful. These active proteins are inactivated by a usual concentration / desalting method or are lost by adsorption, and a sufficient recovery cannot be expected. Typical examples of such proteins include cytokines such as interferon, colony-stimulating factor, growth hormone, growth hormone releasing factor, insulin-like growth factor, plasminogen activator, erythropoietin and interleukin 2. it can.
上記の生理活性たんぱく質は、直接生体組織から抽出
したもの、細胞培養技術あるいは遺伝子組換え技術によ
って製造されたものであってもよい。The above-mentioned physiologically active protein may be one directly extracted from a living tissue, or one produced by a cell culture technique or a gene recombination technique.
透析または限外濾過は、当該技術分野で機知の方法に
よって行なわれる。透析膜は、通常、このような生理活
性たんぱく質の透析に用いられる透析膜を使用して、常
法通り行うことができる。また、限外濾過法は、限外濾
過膜として膜状、平膜状、ホローファイバー状等の形状
を持った限外濾過装置が知られているが、いずれの型の
装置を用いても本発明方法を実施することができる。膜
の材質は、ポリアクリロニトリル系やポリスルホン系
等、種々ある膜のいずれであってもよい。Dialysis or ultrafiltration is performed by methods known in the art. As a dialysis membrane, a dialysis membrane usually used for dialysis of such physiologically active protein can be used in a conventional manner. Further, the ultrafiltration method is known as an ultrafiltration device having a shape such as a membrane, a flat membrane, or a hollow fiber as an ultrafiltration membrane. The inventive method can be carried out. The material of the membrane may be any of various membranes such as polyacrylonitrile-based and polysulfone-based.
［発明の効果］ 本発明によれば、通常の方法によっては活性たんぱく
質の失活や損失を免れ得ないような生理活性たんぱく質
含有水溶液を効率よく濃縮・脱塩することができ、貴重
な生理活性たんぱく質を高濃度に含有する溶液を得るこ
とができる。従って、本発明は、様々な生理活性たんぱ
く質の研究、および臨床への応用の両面での需要に応え
てそれらの活性たんぱく質を提供するという点から、極
めて有益な方法を提供するものである。ADVANTAGES OF THE INVENTION According to the present invention, an aqueous solution containing a physiologically active protein that cannot avoid inactivation or loss of the active protein by an ordinary method can be efficiently concentrated and desalted, and a valuable physiological activity can be obtained. A solution containing a high concentration of protein can be obtained. Therefore, the present invention provides a very useful method from the viewpoint of providing various active proteins in response to the demands for both research and clinical application of various active proteins.
いかに実施例を挙げ、本発明をさらに詳しく説明す
る。The present invention will be described in more detail by way of examples.
実施例１ α−インターフェロンを含む0.01M−トリス、グリシ
ンバッファ−（0.15M、NaCl含有、pH7）200mlを塩酸酸
性にpH調整した後、２％アテロコラーゲンを0.01％（w/
v）になるように添加し、撹拌溶解後、分画分子量10,00
0のポリスルホン系平膜を使って濃縮・脱塩操作を行な
った。なお、脱塩はNaCl濃度が最初の1/1000になるまで
塩酸酸性蒸留水で２倍濃縮、２倍希釈を繰り返した。Example 1 200 ml of 0.01 M Tris containing α-interferon and glycine buffer (0.15 M, containing NaCl, pH 7) was adjusted to pH with hydrochloric acid, and then 2% atelocollagen was added at 0.01% (w / w).
v)), dissolve with stirring, and then cut off the molecular weight of 10,00
Concentration and desalting operations were performed using a 0 polysulfone-based flat membrane. The desalting was carried out by repeating 2-fold concentration and 2-fold dilution with hydrochloric acid-distilled water until the NaCl concentration reached 1/1000 of the initial concentration.
この操作でのα−インターフェロン（IFN）の回収率
をコラーゲンの有無で比較した表１を次に示す。Table 1 below shows a comparison of the recovery rate of α-interferon (IFN) in this operation with and without collagen.
コラーゲンの添加により明らかな回収率の向上が認め
られた。It was recognized that the recovery rate was clearly improved by the addition of collagen.
実施例２ α−インターフェロンを含む0.01M−トリス、グリシ
ンバッファー（0.15M、NaCl含有、pH7）269mlを塩酸酸
性にpH調整し、２％アテロコラーゲンを0.06％（w/v）
になるように添加し、撹拌溶解後、分画分子量10,000の
透析チューブ（ビスキング社製）に入れ10lの塩酸酸性
蒸留水に対して、３時間、３回透析した。この操作での
α−インターフェロン（IFN）の回収率を表２に示す。 Example 2 269 ml of 0.01 M Tris containing α-interferon and glycine buffer (0.15 M, containing NaCl, pH 7) was adjusted to acidic pH with hydrochloric acid, and 2% atelocollagen was 0.06% (w / v).
The resulting solution was added to the above mixture, dissolved with stirring, placed in a dialysis tube with a molecular weight cut off of 10,000 (manufactured by Visking) and dialyzed against 10 liters of hydrochloric acid-distilled water for 3 hours and 3 times. Table 2 shows the recovery rate of α-interferon (IFN) by this operation.
この結果、回収率は非常に高いものであった。 As a result, the recovery rate was extremely high.
実施例３ α−インターフェロンを含む0.01M−トリス、グリシ
ンバッファー（0.15M、NaCl含有、pH7）300mlをヒト血
清アルブミン450mgを撹拌溶解後、塩酸酸性にpH調整
し、２％アテロコラーゲン水溶液3.0mlを加え撹拌溶解
した。この液を分画分子量10,000のポリスルホン系平膜
を使って100mlになるまで濃縮し、NaCl濃度が最初の1/1
000になるまで連続的に塩酸酸性蒸留水を供給しながら
脱塩した。 Example 3 300 ml of 0.01 M Tris containing α-interferon and glycine buffer (0.15 M, containing NaCl, pH 7) was dissolved by stirring 450 mg of human serum albumin with stirring, the pH was adjusted to acidic with hydrochloric acid, and 3.0 ml of 2% atelocollagen aqueous solution was added. It was dissolved with stirring. This solution was concentrated to 100 ml using a polysulfone flat membrane with a cut-off molecular weight of 10,000, and the NaCl concentration was 1/1
Desalination was carried out while continuously supplying hydrochloric acid-distilled water until 000 was reached.
この操作でのα−インターフェロン（IFN）の回収率
を表３に示す。Table 3 shows the recovery rate of α-interferon (IFN) by this operation.
この結果からコラーゲンとヒト血清アルブミンを添加
しても回収率を高めることがわかった。From this result, it was found that the recovery rate was increased even when collagen and human serum albumin were added.
以上実施例を挙げて説明してきたが、本発明はこれら
に限定されるものではなく、生理活性物質の使用目的や
生理活性物質の存在する溶液に適したコラーゲンを選択
すればよい。コラーゲンはpH値や、塩濃度によって容易
に生理活性物質と分離でき、又修飾コラーゲンを用いる
ことにより濃縮や脱塩時のpH値を生理活性物質にとって
望ましい値に設定できる（例えば中性付近であればサク
シニル化コラーゲン）。 Although examples have been described above, the present invention is not limited to these, and a collagen suitable for the purpose of use of the physiologically active substance and the solution in which the physiologically active substance is present may be selected. Collagen can be easily separated from physiologically active substances depending on the pH value and salt concentration, and by using modified collagen, the pH value at the time of concentration and desalting can be set to a desired value for the physiologically active substance (e.g., near neutral). Succinylated collagen).
フロントページの続き (72)発明者 佐々木 慶雄 大阪府茨木市蔵垣内１丁目３番45号 住 友製薬株式会社内 (56)参考文献 特開 昭60−155137（ＪＰ，Ａ) 特開 昭60−41615（ＪＰ，Ａ) 特開 昭59−210027（ＪＰ，Ａ) 特開 昭60−174726（ＪＰ，Ａ) 特開 昭60−228422（ＪＰ，Ａ) 特開 昭62−230729（ＪＰ，Ａ)Front Page Continuation (72) Inventor Yoshio Sasaki 1-345 Kuragakiuchi, Ibaraki City, Osaka Sumitomo Pharmaceutical Co., Ltd. (56) Reference JP-A-60-155137 (JP, A) JP-A-60-41615 (JP, A) JP 59-210027 (JP, A) JP 60-174726 (JP, A) JP 60-228422 (JP, A) JP 62-230729 (JP, A)
Claims (4)
水溶液に、コラーゲンを加え、得られた混合物を限外濾
過または透析することを特徴とする生理活性たんぱく質
水溶液の濃縮・脱塩方法。1. A method for concentrating and desalting an aqueous physiologically active protein solution, which comprises adding collagen to an aqueous solution containing a purified biologically active protein and subjecting the resulting mixture to ultrafiltration or dialysis.
モル比で約10-4以上の割合で加えることを特徴とする特
許請求の範囲第１項に記載の方法。2. Collagen for physiologically active protein,
The method according to claim 1, wherein the method is added in a molar ratio of about 10 -4 or more.
を特徴とする特許請求の範囲第１−２項のいずれかに記
載の方法。3. The method according to any one of claims 1-2, wherein the collagen is atelocollagen.
コロニー形成刺激因子、成長ホルモン、成長ホルモン放
出因子、インシュリン様成長因子、プラスミノーゲン活
性化因子、エリスロポエチン、インターロイキン２等の
サイトカインであることを特徴とする特許請求の範囲第
３項記載の方法。4. A bioactive protein is interferon,
The method according to claim 3, which is a cytokine such as colony-stimulating factor, growth hormone, growth hormone-releasing factor, insulin-like growth factor, plasminogen activator, erythropoietin, and interleukin-2. .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62334046A JP2537935B2 (en) | 1987-12-29 | 1987-12-29 | Agricultural reduction and desalination method for physiologically active substances |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62334046A JP2537935B2 (en) | 1987-12-29 | 1987-12-29 | Agricultural reduction and desalination method for physiologically active substances |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01175995A JPH01175995A (en) | 1989-07-12 |
| JP2537935B2 true JP2537935B2 (en) | 1996-09-25 |
Family
ID=18272904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62334046A Expired - Fee Related JP2537935B2 (en) | 1987-12-29 | 1987-12-29 | Agricultural reduction and desalination method for physiologically active substances |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2537935B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115232204B (en) * | 2022-08-10 | 2024-09-27 | 成都奇璞生物科技有限公司 | Collagen and desalting process, concentrating process, preparation process, application and filler of sterile and crosslinked collagen |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59210027A (en) * | 1983-05-14 | 1984-11-28 | Kagaku Gijutsucho Hoshasen Igaku Sogo Kenkyusho | Production of cerebrospinal fluid (csf) |
| JPS6041615A (en) * | 1983-08-12 | 1985-03-05 | Green Cross Corp:The | Preparation of colony stimulating factor |
| JPS60155137A (en) * | 1984-01-23 | 1985-08-15 | Takeda Chem Ind Ltd | Preparation of aqueous solution of human gamma type interferon having high concentration |
| JPS60174726A (en) * | 1984-02-21 | 1985-09-09 | Nippon Shinyaku Co Ltd | Pharmaceutical composition for injection |
| JPS60228422A (en) * | 1984-04-26 | 1985-11-13 | Suntory Ltd | Stabilized preparation of physiologically active substance |
| JPH0725688B2 (en) * | 1986-03-31 | 1995-03-22 | 住友製薬株式会社 | CSF sustained release formulation |
-
1987
- 1987-12-29 JP JP62334046A patent/JP2537935B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01175995A (en) | 1989-07-12 |
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