JP2012503596A5

JP2012503596A5 -

Info

Publication number: JP2012503596A5
Application number: JP2011527187A
Authority: JP
Filing date: 2009-05-26
Publication date: 2013-10-17

Description

本発明の第二の態様は、糖タンパク質ホルモンにおけるウイルスを除去及び／或いは不活性化する方法であって、
（ａ）糖タンパク質に対し、沈殿形成及び／或いは懸濁液攪拌処理から選ばれる有機溶液処理を行う工程と、
（ｂ）糖タンパク質に対し、微孔膜によるろ過を行う工程と、
（ｃ）糖タンパク質に対し、イオン交換クロマトグラフィーを行う工程と、
からなる群から選ばれた一つまたは複数の工程を、任意に交換可能な順序で含む方法を提供する。 A second aspect of the present invention is a method for removing and / or inactivating viruses in glycoprotein hormones, comprising:
(A) a step of subjecting the glycoprotein to organic solution treatment selected from precipitation formation and / or suspension stirring treatment;
(B) a step of filtering the glycoprotein with a microporous membrane;
(C) performing ion exchange chromatography on the glycoprotein;
A method comprising one or more steps selected from the group consisting of, optionally in exchangeable order.

もう一つの好ましい例において、工程（ａ）では、糖タンパク質を零下20℃−20℃で有機溶液にて１分−4時間処理する。
もう一つの好ましい例において、工程（ｂ）では、前記微孔膜は、孔径が１０００Ｄ以上の限外ろ過膜、孔径が１−１００ｎｍのウイルス除去膜、及び孔径が０．１−１μｍの細菌ろ過膜から選ばれるものである。
もう一つの好ましい例において、工程（ｂ）では、前記微孔膜は、孔径が１０００００Ｄ以上の限外ろ過膜、孔径が１０−８０ｎｍのウイルス除去膜、及び孔径が０．２−０．８μｍの細菌ろ過膜から選ばれるものである。 In another preferred example, in step (a), the glycoprotein is treated at 20 ° C.-20 ° C. with an organic solution for 1 minute-4 hours under zero.
In another preferred example, in the step (b), the microporous membrane is an ultrafiltration membrane having a pore size of 1000 D or more, a virus removal membrane having a pore size of 1-100 nm, and a bacterium having a pore size of 0.1-1 μm. It is selected from filtration membranes.
In another preferred example, in the step (b), the microporous membrane is an ultrafiltration membrane having a pore size of 100,000 D or more, a virus removal membrane having a pore size of 10-80 nm, and a pore size of 0.2-0.8 μm. Selected from bacterial filtration membranes.

具体的な実施形態
従来技術にはタンパク質におけるウイルスを除去及び／或いは不活性化する方法がいくつかあるが、同様の方法でも、異なるタンパク質に対して、予想できない結果が出てしまい、逆効果が出ることもある。ある方法は、あるタンパク質に対しては実施可能であっても、具体的な実施条件が他のタンパク質に比べて大きな差がある。この事情に鑑み、発明者らは幅広く深く研究したところ、驚くことに、有機溶液処理、微孔膜ろ過およびイオン交換クロマトグラフィーのうちの一つ又は全ての工程を任意に組み合わせることで、グリコシル化タンパク質におけるウイルスを除去／不活性化するという目的を達成することができ、これにより、ウイルスは緩やかな条件で除去／不活性化されるとともに、目的物のグリコシル化タンパク質の活性は保存される、ことを見出した。この知見に基づき、発明者らは本発明を完成した。 Specific Embodiments Although there are several methods in the prior art for removing and / or inactivating viruses in proteins, similar methods can produce unpredictable results for different proteins and have an adverse effect. May come out. Although some methods can be performed on certain proteins, the specific implementation conditions differ greatly compared to other proteins. In view of this situation, the inventors have conducted extensive and extensive research, and surprisingly, glycosylation can be achieved by arbitrarily combining one or all of the steps of organic solution processing, microporous membrane filtration, and ion exchange chromatography. The goal of removing / inactivating the virus in the protein can be achieved, whereby the virus is removed / inactivated under mild conditions and the activity of the glycosylated protein of interest is preserved, I found out. Based on this finding, the inventors completed the present invention.

探索している間に、発明者らは、エタノールやアセトンなどの有機溶媒による処理は、ウイルスに対しては不活性化作用があるが、ＨＣＧ、ＨＭＧ、ＦＳＨ、ＬＨの活性に対してはなんらの影響もない、ことを気付いた。さらに、適切な微孔膜ろ過技術によれば、ウイルス粒子を阻止することができるだけでなく、目的物を膜に透過させることも可能で、目的物とウイルスを分離する目的を達成できる、ことも見出した。また、発明者らは、試験中で意外なことに、条件を適切に制御すれば、イオン交換クロマトグラフィーにより目的物を吸着することなくウイルスをイオン樹脂に吸着することができ、目的物からウイルスを除去する目的を達成できる、ことを見出した。
前記の三つの工程のうちの一つ又は複数の工程を任意に組み合わせることで、ウイルスを効率的に除去／不活性化することができ、安全で信頼的な製品が得られる。 During the search, the inventors have found that treatment with an organic solvent such as ethanol or acetone has an inactivating effect on the virus, but no activity on the activity of HCG, HMG, FSH, or LH. I noticed that there was no influence. Furthermore, according to appropriate microporous membrane filtration technology, not only can the virus particles be blocked, but also the target can be permeated through the membrane, and the purpose of separating the target from the virus can be achieved. I found it. In addition, the inventors surprisingly, in the test, if the conditions are appropriately controlled, the virus can be adsorbed to the ion resin without adsorbing the target product by ion exchange chromatography. It has been found that the purpose of removing can be achieved.
By arbitrarily combining one or more of the three steps, the virus can be efficiently removed / inactivated, and a safe and reliable product can be obtained.

方法
本発明により提供される糖タンパク質におけるウイルスを除去及び／或いは不活性化する方法は一つまたは複数の工程からなり、複数の工程の順序が任意に組み合わせることができる。
前記工程は、有機溶液処理、微孔膜処理、及びイオン交換クロマトグラフィー処理である。 Method The method for removing and / or inactivating viruses in glycoproteins provided by the present invention comprises one or more steps, and the order of the steps can be arbitrarily combined.
The steps are organic solution treatment, microporous membrane treatment, and ion exchange chromatography treatment.

前記微孔膜処理は、糖タンパク質の濃度１５−６０ミリグラム／ミリリットル（好ましくは２０−４０ミリグラム／ミリリットル）の溶液を微孔膜に透過させて、ウイルスを除去及び／或いは不活性化するものである。前記微孔膜は、孔径が１０００Ｄ以上の限外ろ過膜、孔径が１−１００ｎｍのウイルス除去膜、及び孔径が０．１−１μｍの細菌ろ過膜から選ばれるもので、好ましくは孔径が１０００００Ｄ以上の限外ろ過膜、孔径が１０−８０ｎｍのウイルス除去膜、及び孔径が０．３−０．８μｍの細菌ろ過膜から選ばれるものである。前記溶液は、ｐＨ６−１０で、好ましくはｐＨ７−９であり、例えばリン酸塩、酢酸塩またはＴｒｉｓ緩衝液である。 The microporous membrane treatment removes and / or inactivates viruses by allowing a glycoprotein concentration of 15-60 mg / milliliter (preferably 20-40 mg / milliliter) to permeate the microporous membrane. is there. The microporous membrane is selected from an ultrafiltration membrane having a pore size of 1000 D or more, a virus removal membrane having a pore size of 1-100 nm, and a bacterial filtration membrane having a pore size of 0.1-1 μm, and preferably has a pore size of 100,000. It is selected from an ultrafiltration membrane of D or more, a virus removal membrane having a pore size of 10-80 nm, and a bacterial filtration membrane having a pore size of 0.3-0.8 μm. The solution is pH 6-10, preferably pH 7-9, such as phosphate, acetate or Tris buffer.

Claims

(A) a step of performing an organic solution treatment to inactivate viruses by mixing a glycoprotein and an organic solution to form a precipitate or mixing and stirring to form a suspension ;
(B) a step of filtering the glycoprotein with a microporous membrane ;
(C) glycoprotein hand, and performing ion exchange chromatography,
Seen including,
In the step (a), the organic solution is a 70-100 v / v% organic solvent / water solution, and the organic solution is selected from ethanol, methanol, acetone, and diethyl ether, and the glycoprotein and organic The weight: volume ratio of the solution is 0.5-2 grams: 15-60 ml,
In the step (b), the microporous membrane is selected from an ultrafiltration membrane having a pore size of 1000 D or more, a virus removal membrane having a pore size of 1-100 nm, and a bacterial filtration membrane having a pore size of 0.1-1 μm. Yes,
In step (c), DEAE Sephadex is used as an ion exchange resin in ion exchange chromatography,
A method for removing and / or inactivating a virus in a glycoprotein hormone, wherein the glycoprotein hormone is selected from chorionic gonadotropin, follicle stimulating hormone, and menopausal gonadotropin .

The method according to claim 1, wherein the organic solution is selected from ethanol and acetone.

The method according to claim 1, wherein the weight ratio of the glycoprotein and the organic solution is 0.7-1.5 gram: 20-40 milliliters.

The method according to claim 1, wherein in step (a), the glycoprotein is treated with an organic solution at a temperature of 3 ° C to 3 ° C under zero for 0.5 minutes to 6 hours.

The method according to claim 1, wherein in step (a), the glycoprotein is treated with an organic solution at 0 ° C to 2 ° C under zero for 1 minute to 4 hours.

In the step (b), the microporous membrane is selected from an ultrafiltration membrane having a pore size of 100,000 D or more, a virus removal membrane having a pore size of 10-80 nm, and a bacterial filtration membrane having a pore size of 0.2-0.8 μm. The method according to claim 1, characterized in that:

The method according to claim 1, wherein in the step (c), the ion exchange chromatography is performed under conditions of pH 5-10.

[8] The method according to claim 7, wherein in the step (c), the ion exchange chromatography is performed under conditions of pH 6-8.