제1도는 본 발명에 따른 정제 방법의 개요를 도표로 나타낸 것이고,1 is a diagram showing an overview of the purification method according to the invention,
제2도는 본 발명에 따라 최종 정제된 인간 인터페론 베타를 15% SDS-PAGE 및 RP-HPLC한 결과를 나타낸 것이다.Figure 2 shows the results of 15% SDS-PAGE and RP-HPLC of the final purified human interferon beta according to the present invention.
본 발명은 인간 인터페론 베타의 정제방법에 관한 것으로, 보다 상세하게는 유전공학적인 방법에 의해 대장균에서 대량 발현된 순도에 낮은 인간 인터페론 베타를 고순도의 인간 인터페론 베타로 정제하는 방법에 관한 것이다.The present invention relates to a method for purifying human interferon beta, and more particularly, to a method for purifying human interferon beta having high purity expressed in Escherichia coli with high purity human interferon beta by genetic engineering method.
일반적으로 인터페론은 세포의 여러기능을 조절해주는 작용을 갖고 있는 단백질의 한종류로서 그 기능이 매우 다양한 바, 예를들면, 바이러스로부터의 세포보호, 조직배양에서나 골수에서의 세포분열 억제, T세포작용의 조절, 자연면역세포의 기능항진 유도에 의한 식균작용 상승, 또는 특수 암세포의 분열억제 등의 기능이 있는 것으로 알려져 있으며, 특히 항바이러스제와 항암제로서 유용하다. 이들 인터페론은 α-, β-, γ-인터페론 및 그 밖의 다양한 종류의 인터페론이 알려져 있는데, 이중 인터페론 베타는 분자량 약 23,000의 당 단백질로서(Utsumi, Jun, J.Biochem., 101, 1199(1987))피부암, 뇌종양, 다발성 경화증 및 바이러스성 B형 간염 치료제로 효과가 있음이 알려져 있다.In general, interferon is a kind of protein that has a function of regulating various functions of cells, and its functions vary widely, for example, cell protection from viruses, cell division in tissue culture or bone marrow, and T-cell action. It is known to have a function such as regulation of phagocytosis, increase of phagocytosis by induction of hyperfunction of natural immune cells, or suppression of division of special cancer cells. These interferons are known to be α-, β-, γ-interferon and various other types of interferons, among which interferon beta is a sugar protein having a molecular weight of about 23,000 (Utsumi, Jun, J. Biochem., 101, 1199 (1987) It is known to be effective in treating skin cancer, brain tumor, multiple sclerosis and viral hepatitis B.
인간 인터페론 베타를 얻기 위하여 종래에는 사람의 섬유아세포를 대량으로 키우면서 폴리 I,폴리 C 및 싸이클로 헥사미드로 슈퍼인덕션시키는 방법을 사용하였으나, 최근에는 유전공학 기술을 이용하여 대장균에서 인간 인터페론 베타를 발현시켜 대량으로 얻을 수 있게 되었다(대한민국 공개특허 제87-11248호).Conventionally, in order to obtain human interferon beta, a method of superinduction with poly I, poly C and cyclo hexamid while growing human fibroblasts has been used. However, recently, human interferon beta is expressed in E. coli using genetic engineering technology. A large amount can be obtained (Korean Patent Publication No. 87-11248).
유전공학적인 방법으로 대장균에서 발현된 인간 인터페론 베타를 정제하는 종래 방법으로는 소수성이 강한 인간 인터페론 베타를 소디움 도데실 설페이트를 사용하여 용해시킨 후 원심분리하고 용매로 처리하여 컬럼 크로마토그라피를 통과시키는 방법을 이용하여 왔으나, 이 방법으로는 엔도톡신을 완전히 제거하기가 어렵고 또한 소디움 도데실 설페이트가 완전히 제거되지 않아 임상에 적합하지 않은것 등이 문제점으로 지적되어 왔다.In the conventional method of purifying human interferon beta expressed in E. coli by genetic engineering method, hydrophobic human interferon beta is dissolved using sodium dodecyl sulfate, centrifuged and treated with solvent to pass column chromatography. However, it has been pointed out that this method is difficult to completely remove the endotoxin, and that sodium dodecyl sulfate is not completely removed and is not suitable for clinical use.
특히 대장균에서 발현된 인간 인터페론 베타의 경우 단단한 봉입체를 형성하므로 이를 완전히 용해시키기 위하여 베타 머켑토에탄올, 디티오트레이톨 등과 같은 환원제와 소디움 도데실 설페이트, 염산 구아니딘, 우레아 등과 같은 불활성물질을 사용하여 왔는데, 이 경우 인간 인터페론 베타의 3차 구조는 인체 내에서 존재하는 인터페론 베타의 3차 구조와 상이하여 생리활성도가 떨어지는 문제점이 있었다.In particular, human interferon beta expressed in Escherichia coli forms a hard inclusion body, so in order to completely dissolve it, reducing agents such as beta merethantoethanol and dithiothritol, and inerts such as sodium dodecyl sulfate, guanidine hydrochloride, and urea have been used. In this case, the tertiary structure of human interferon beta is different from the tertiary structure of interferon beta present in the human body, and thus has a problem of inferior physiological activity.
이에 본 발명자들은 상기와 같은 문제점들을 해결하고자 연구 노력한 결과, 인간 인터페론 베타를 정제하는데 있어서 세포파괴 및 용해 단백질 제거단계를 거쳐 비이온성 계면활성제 추출법, 적당한 구아디닌 농도에서의 산화, 역상 크로마토그라법, 이온교환 크로마토그라피법을 적용하여 생체내에서 높은 생리활성도를 갖는 높은 순도의 인간 인터페론 베타를 대량 생산하는데 유용하게 이용될 수 있는 인간 인터페론 베타의 정제 방법을 완성하게 되었다.Accordingly, the present inventors have made efforts to solve the above problems, and as a result, in the purification of human interferon beta, the process of cell destruction and removal of soluble proteins, followed by extraction of nonionic surfactant, oxidation at appropriate guadinin concentration, and reverse phase chromatography By applying ion exchange chromatography, a method of purifying human interferon beta that can be usefully used for mass production of high purity human interferon beta having high physiological activity in vivo has been completed.
본 발명의 목적은 유전공학적인 방법에 의해 대장균에서 대량 발련된 인간 인터페론 베타를 비이온성 계면활성제 추출법, 염산구아니딘으로의 산화, 역상 크로마토그라피와 양이온 교한 크로마토그라피 등을 이용하여 높은 생리활성을 갖는 고순도의 인간 인터페론 베타로 정제시키는 방법을 제공하는데 있다.An object of the present invention is a high purity having high physiological activity using human interferon beta mass-developed in E. coli by genetic engineering method, nonionic surfactant extraction method, oxidation to guanidine hydrochloride, reverse phase chromatography and cation exchange chromatography, etc. To provide a method for purifying with human interferon beta.
이하 본 발명을 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail.
본 발명은 유전공학적인 방법으로 대장균에서 발현된 인간 인터페론 베타를 분리 정제하는데 있어서, 인간 인터페론 베타가 발현되어 있는 대장균을 완충용액에 현탁 및 분쇄시킨 후 원심분리하여 용해 단백질을 제거하고 얻어진 불용해 침전물을 용해제인 비이온성 계면활성제가 함유되어 있는 염산 구아니딘에 용해시킨 다음 용해된 단백질을 초산완충용액에 대해 투석하고 원심분리하여 얻은 상등액을 젤 여과 크로마토그라피하고 적당한 농도의 염산 구아니딘 용액에서 산화시킨 다음 역상 크로마토그라피와 양이온 교환 크로마토그라피를 순차적으로 실시하여서 되는 것임을 특징으로 하는 고순도 및 생리활성도가 높은 인간 인터페론 베타의 정제방법이다.In the present invention, in the separation and purification of human interferon beta expressed in E. coli by genetic engineering method, the insoluble precipitate obtained by suspending and pulverizing E. coli expressing human interferon beta in a buffer solution and then centrifuging to remove soluble protein Was dissolved in guanidine hydrochloride containing a nonionic surfactant as a solubilizer, and the supernatant obtained by dialysis and centrifugation of the dissolved protein in acetic acid buffer solution was subjected to gel filtration chromatography and oxidized in a solution of guanidine hydrochloride at an appropriate concentration. It is a method for purifying human interferon beta having high purity and high physiological activity, characterized in that the chromatography and cation exchange chromatography are performed sequentially.
이하 본 발명을 더욱 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in more detail.
인간 인터페론 베타가 발현되어 있는 대장균을 완충용액 [20mM 트리스(트리스-히드록시메틸아미노에탄, 500mM EDTA]에 현탁시킨 후 라이소자임을 첨가하여 세포 균질기로 파괴시킨다. 이를 원심분리하여 용해 단백질을 제거한 후 얻어진 불용해 침전물을 요소가 함유되어 있는 완충용액에 현탁시켜 이를 원심분리하고 얻어진 불용해 침전물을 염산구아니딘이 함유되어 있는 완충용액에 현탁시켜 원심분리하여 불용해 침전물을 얻는다.E. coli expressing human interferon beta is suspended in a buffer solution [20 mM Tris (tris-hydroxymethylaminoethane, 500 mM EDTA), and then lysozyme is added and destroyed by a cell homogenizer. Insoluble precipitate is suspended in a buffer solution containing urea and centrifuged, and the obtained insoluble precipitate is suspended in a buffer solution containing guanidine hydrochloride to centrifugation to obtain an insoluble precipitate.
상기에서 얻어진 침전물을 2% 트윈류 또는 2% 트리톤류가 함유되어 있는 8M 염산구아니딘에 재용해시킨 후 초산 완충용액(20mM 초산,1mM EDTA, 50mM KCI, 1% 덱스트로즈)에 대해 투석시켜 염산 구아니딘을 완전히 제거한 다음 원심분리하여 상등액을 얻는다.The precipitate obtained above was redissolved in 8M guanidine hydrochloride containing 2% twins or 2% tritons, and then dialyzed against acetic acid buffer solution (20 mM acetic acid, 1 mM EDTA, 50 mM KCI, 1% dextrose) to hydrochloric acid. Guanidine is completely removed and then centrifuged to obtain supernatant.
이렇게 하여 얻어진 상등액을 배제한계가 25,000이상 또는 30,000이상인 겔 여과 크로마토그라피를 적용하여 단백질을 회수하고 회수한 단백질 용액에 염산구아니딘의 농도가 6M이 되도록 염산 구아니딘을 첨가한 후 50mM 트리스 완충용액으로 3M의 염산구아니딘이 되게 희석하여 pH를 7~9로 맞춘 뒤 산소 존재하에 염산구아니딘에 의해 산화되도록 10분~2시간 동안 방치한 후 초산완충용액(20mM 초산, pH 4.5)에 대해 투석시켜 산화반응을 종결시킨다.The supernatant thus obtained was subjected to gel filtration chromatography with an exclusion limit of 25,000 or more and 30,000 or more, to recover the protein, and to the recovered protein solution, guanidine hydrochloride was added so that the concentration of guanidine hydrochloride was 6M. Dilute to guanidine hydrochloride to adjust the pH to 7-9, and leave it for 10 minutes to 2 hours to oxidize with guanidine hydrochloride in the presence of oxygen, then terminate the oxidation reaction by dialysis against acetic acid buffer solution (20mM acetic acid, pH 4.5). Let's do it.
상기에서 얻어진 단백질을 역상 크로마토그라피(Matrex Silica SC.Amicon사)에 흡착시킨 뒤 에탄올, 아세토니트릴, 프로판올 등의 용매로 용출시키고 양이온 교환 크로마토그라피에 적용시키게 되는데 이때 젤로는 CM-세파로즈 CL-6B(Pharmacia사) 또는 S-세파로즈(Pharmacia사)등을 사용할 수 있다.The protein obtained above is adsorbed onto reverse phase chromatography (Matrex Silica SC. Amicon), eluted with a solvent such as ethanol, acetonitrile, propanol, and subjected to cation exchange chromatography, wherein the gel is CM-Sepharose CL-6B. (Pharmacia) or S-Sepharose (Pharmacia) etc. can be used.
상기 컬럼을 통과시켜 용출된 각 분획을 전기영동하여서 인간 인터페론 베타의 순도가 98% 이상되는 부분을 모음으로써 높은 생리활성을 갖는 인간 인터페론 베타를 고순도로 정제할 수 있게 된다.Electrophoresis of each fraction eluted through the column allows the human interferon beta having high physiological activity to be purified with high purity by collecting portions of 98% or more purity of human interferon beta.
본 출원인은 본 발명의 인간 인터페론 베타의 생산능을 가진 미생물을 Luck-β-IFN-1E라 명명하고 이를 1990년 12월 12일에 재단법인 한국종균협회에 기탁번호 KFCC-10717로 기탁하였다.Applicant named Luck-β-IFN-1E as a microorganism having a production capacity of human interferon beta of the present invention and deposited it on December 12, 1990 with the Accession No. KFCC-10717 to the Korea spawn association.
이하, 본 발명을 실시예에 의거하여 상세히 설명하면 다음과 같다.Hereinafter, the present invention will be described in detail with reference to Examples.
[실시예 1]Example 1
[단계 1 ; 세포파괴 및 용해 단백질의 제거][Step 1; Cell destruction and removal of lysed protein]
인간 인터페론 베타가 발현된 대장균 1㎏을 2.8ℓ완충용액(20mM 트리스, 50mM EDTA, pH 7.8)에 현탁시킨 후 1㎎의 라이소자임을 첨가하여 상온에서 1~2시간 방치한 후 얼음조에서 약 5분~10분 간격으로 초음파 세포파괴하였다. 그 다음, 고속 원심분리기(Beckman사)를 사용하여 약 20,000xg에서 30분간 원심분리하여 용해 단백질을 제거하고 침전물을 2.8ℓ의 1M 요소에 현탁시켜 약 10분간 저어준 다음 다시 20,000xg에서 30분간 원심분리하여 상등액(용해 단백질)을 제거하고 침전물을 2.8ℓ의 1M 염산구아니딘에 현탁시킨 10분간 저어주었다. 그 다음 현탁용액을 약 20,000xg에서 30분간 원심분리하여 얻은 침전물을 단계 2에서 사용하였다.Suspension of 1 kg of E. coli expressing human interferon beta in 2.8ℓ buffer solution (20mM Tris, 50mM EDTA, pH 7.8) and add 1mg of lysozyme and leave at room temperature for 1 ~ 2 hours Ultrasonic cell destruction was performed at intervals of ˜10 minutes. Then, using a high speed centrifuge (Beckman), centrifuged at about 20,000xg for 30 minutes to remove lysed protein, the precipitate was suspended in 2.8 L of 1M urea, stirred for about 10 minutes, and then centrifuged at 20,000xg for 30 minutes. The supernatant (dissolved protein) was removed and the precipitate was stirred for 10 minutes in 2.8 L of 1M guanidine hydrochloride. Then, the precipitate obtained by centrifuging the suspension solution at about 20,000 × g for 30 minutes was used in step 2.
[단계 2 ; 비이온성 계면활성제를 이용한 인간 인터페론 베타의 추출][Step 2; Extraction of Human Interferon Beta Using Nonionic Surfactants]
상기 단계 1에서 얻어진 침전물을 800㎖의 8M 염산구아니딘에 넣어 4~5시간 동안 용해시킨 다음 β-머캡토에탄올을 50mM 되게 넣은 후 60℃에서 10분간 방치하여 완전 환원시킨 뒤 약 20,000xg에서 40분간 원심분리하여 용해되지 않은 부분을 제거하고 상등액을 얻었다. 8M 염산 구아니딘 상등액에 트윈 80을 2% 농도되게 첨가한 후 5mM EDTA, 50mM 염화칼륨, 1% 덱스트로즈를 함유한 20mM 초산 완충용액(pH 4.5)에 대해 약 5,000배 투석한 다음 20,000xg에서 30분간 원심분리하여 얻은 상등액을 15% 소디움 도데실 설페이트-폴리아크릴 아마이드 겔 전기영동한 결과, 순도가 85% 이상인 인간 인터페론 베타를 얻을 수 있었다.The precipitate obtained in step 1 was dissolved in 800 ml of 8M guanidine hydrochloride for 4-5 hours, and then β-mercaptoethanol was added to 50 mM and left at 60 ° C. for 10 minutes for complete reduction, followed by 40 minutes at about 20,000 × g. The undissolved portion was removed by centrifugation to obtain a supernatant. After adding 2% of Tween 80 to the 8M guanidine supernatant, dialyzed about 5,000 times in 20mM acetic acid buffer solution (pH 4.5) containing 5mM EDTA, 50mM potassium chloride and 1% dextrose, followed by 30 minutes at 20,000xg. The supernatant obtained by centrifugation was subjected to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain human interferon beta having a purity of 85% or more.
[단계 3 : 인간 인터페론 베타의 산화][Step 3: Oxidation of Human Interferon Beta]
단계 2에서 얻은 상등액의 염 및 환원제를 제거하기 위하여 5mM 수산화나트륨(pH 10.5)으로 미리 평형시킨 세파덱스 G-25(Pharmacia사) 컬럼(10×100㎝)에 통과시켜 단백질 부분을 회수하였다. 회수된 단백질에 염산구아니딘을 첨가하여 6M 농도가 되게 한 다음 50mM 트리스 완충용액(pH 9.5)으로 3M 염산구아니딘이 되도록 희석시킨 뒤 pH를 8.0~9.0으로 조절하여 약 2시간 동안 공기 존재하에 산화반응시켰다.The protein portion was recovered by passing through a Sephadex G-25 (Pharmacia) column (10 x 100 cm) previously equilibrated with 5 mM sodium hydroxide (pH 10.5) to remove the salt and reducing agent of the supernatant obtained in step 2. Guanidine hydrochloride was added to the recovered protein to a concentration of 6M, diluted with 50 mM Tris buffer (pH 9.5) to 3M guanidine hydrochloride, and then the pH was adjusted to 8.0-9.0 to oxidize in the presence of air for about 2 hours. .
산화시킨 용액을 20mM 초산완충용액(pH 4.5)에 대해 투석을 하여 산화반응을 종결시켰다.The oxidized solution was dialyzed against 20 mM acetic acid buffer solution (pH 4.5) to terminate the oxidation reaction.
[단계 4 ; 역상 크로마토그래피에 의한 정제][Step 4; Purification by Reversed Phase Chromatography]
단계 3에서 투석된 용액을 20mM 초산완충용액(pH 4.5)으로 미리 평형시킨 역상 크로마토그라피(Matrex Silica SC, Amicon사) 컬럼(5×10㎝)에 흡착시키고 20mM 초산완충용액(5%는 노말-프로판올, pH4.5) 및 20mM 초산완충용액(70% 노말-프로판올, pH 4.5)을 이용하여 노말-프로판올의 농도구배가 5%~70%까지 되도록 선형구배로 용출시켰으며, 이때의 용출속도는 1㎖/min으로 하였다. 용출된 각 분획을 15% 소디움 도데실 설페이트-폴리아크릴아마이드 겔 전기영동하여 덴시토미터로 스캔닝한 결과, 인간 인터페론 베타의 순도가 90%이상 되는 분획만을 모았다.The solution dialyzed in step 3 was adsorbed onto a reversed phase chromatography (Matrex Silica SC, Amicon) column (5 × 10 cm) previously equilibrated with 20 mM acetic acid buffer solution (pH 4.5) and 20 mM acetic acid buffer solution (5% was normal- Propanol, pH4.5) and 20 mM acetic acid buffer solution (70% normal-propanol, pH 4.5) were used to elute in a linear gradient such that the concentration gradient of normal-propanol was 5% to 70%. It was set to 1 ml / min. Each eluted fraction was scanned with a densitometer by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and only fractions having a purity of 90% or more of human interferon beta were collected.
[단계 5 ; 양이온 교환 수지 크로마토그래피에 의한 정제][Step 5; Purification by Cation Exchange Resin Chromatography]
단계 4에서 얻은 인간 인터페론 베타 분획을 20mM 초산 완충용액(pH 5.3)으로 미리 평형시킨 CM-세파로스 CL-6B(Pharmacia 사) 컬럼(5×10㎝)에 흡착시킨 뒤 20mM 초산완충용액(pH5.3) 및 1M 염화나트륨이 함유된 20mM 초산완충용액(pH 5.3)을 이용하여 염화나트륨의 농도가 구배가 0~1M까지 되도록 선형구배로 용출시켰으며 이때의 용출속도는 1㎖/min으로 하였다. 용출된 각 분획을 15% 소디움 도데실 설페이트-폴리아크릴아미드 겔 전기영동하여 덴시토미터로 스케닝한 결과, 인간 인터페론 베타의 순도가 98% 이상되는 분획을 얻었다. 이렇게 하여 얻어진 인간 인터페론 베타를 소디움 도데실 설페이트-폴리아크릴아미드겔 전기영동한 결과와 RP-HPLC한 결과를 제2도에 나타내었는바, RP-HPLC 선형구배 조건으로 아세토니트릴의 농도가 0~10분까지는 0~45%가 되게, 10~40분까지는 45%~65%가 되게, 40~60분까지는 65%~100%농도가 되도록 하였으며 용출속도는 1㎖/min이었다.The human interferon beta fraction obtained in step 4 was adsorbed onto a CM-Sepharose CL-6B (Pharmacia) column (5 × 10 cm) previously equilibrated with 20 mM acetic acid buffer (pH 5.3), followed by 20 mM acetic acid buffer (pH 5. 3) and 1M sodium chloride containing 20mM acetate buffer solution (pH 5.3) was eluted with a linear gradient so that the concentration of sodium chloride to the gradient 0 ~ 1M, the dissolution rate was set to 1ml / min. Each eluted fraction was 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and screened with a densitometer to obtain a fraction of 98% purity of human interferon beta. The results of electrophoresis of sodium dodecyl sulfate-polyacrylamide gel and the result of RP-HPLC of the human interferon beta thus obtained are shown in FIG. 2. The concentration of acetonitrile under the conditions of RP-HPLC linear gradient is 0-10. The concentration was 0-45%, the concentration was 45% -65% until 10-40 minutes, and the concentration was 65% -100% by 40-60 minutes, and the dissolution rate was 1 ml / min.
[실시예 2]Example 2
[비이온성 계면활성제를 이용한 인간 인터페론 베타의 안정화][Stabilization of Human Interferon Beta Using Nonionic Surfactants]
단계 5에서 얻어진 최종 인간 인터페론 베타에 0.1%~0.2~의 농도로 트윈류 또는 트리톤 X-305 등과 같은 주사제 사용에 적합한 비이온성 계면활성제를 첨가한 후 20g/ℓ 글리신, 1.6g/ℓ 인산나트륨, 100mM 염화나트륨, 1% 덱스트로즈 용액에 투석하여 주사제에 적합한 인간 인터페론 베타를 얻었다. 이렇게하여 얻어진 인간 인터페론 베타의 생리활성도는 5.0×10IU/㎎이었다.20 g / L glycine, 1.6 g / L sodium phosphate, after adding a nonionic surfactant suitable for injection use such as Tween or Triton X-305 at a concentration of 0.1% to 0.2 to the final human interferon beta obtained in step 5, Dialysis in 100 mM sodium chloride, 1% dextrose solution gave human interferon beta suitable for injection. The physiological activity of the human interferon beta thus obtained was 5.0 × 10 IU / mg.
제1도는 상기 본 발명에 따른 정제방법의 개요를 간략하게 도표로 나타낸 것이다.1 is a simplified diagram of the outline of the purification method according to the present invention.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019910006849AKR930006706B1 (en) | 1991-04-29 | 1991-04-29 | Method of Purifying Human Interferon Beta |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1019910006849AKR930006706B1 (en) | 1991-04-29 | 1991-04-29 | Method of Purifying Human Interferon Beta |
| Publication Number | Publication Date |
|---|---|
| KR920019934A KR920019934A (en) | 1992-11-20 |
| KR930006706B1true KR930006706B1 (en) | 1993-07-23 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1019910006849AExpired - Fee RelatedKR930006706B1 (en) | 1991-04-29 | 1991-04-29 | Method of Purifying Human Interferon Beta |
| Country | Link |
|---|---|
| KR (1) | KR930006706B1 (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002034791A3 (en)* | 2000-10-27 | 2003-10-09 | Chiron Corp | Methods of purification and recovery of interferon-beta |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002034791A3 (en)* | 2000-10-27 | 2003-10-09 | Chiron Corp | Methods of purification and recovery of interferon-beta |
| US7544354B2 (en) | 2000-10-27 | 2009-06-09 | Novartis Vaccines And Diagnostics | Methods of protein purification and recovery |
| Publication number | Publication date |
|---|---|
| KR920019934A (en) | 1992-11-20 |
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