KR101504824B1 - Hyperglycosylated long-acting human growth hormone and method for preparing the same - Google Patents
Hyperglycosylated long-acting human growth hormone and method for preparing the same Download PDFInfo
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- KR101504824B1 KR101504824B1 KR1020130063053A KR20130063053A KR101504824B1 KR 101504824 B1 KR101504824 B1 KR 101504824B1 KR 1020130063053 A KR1020130063053 A KR 1020130063053A KR 20130063053 A KR20130063053 A KR 20130063053A KR 101504824 B1 KR101504824 B1 KR 101504824B1
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- South Korea
- Prior art keywords
- growth hormone
- human growth
- nexp
- alpha
- protein
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Abstract
본 발명은 체내 지속성이 증대된 고당화 인간성장호르몬 NexP-hGH 단백질 및 이의 제조방법에 관한 것이다. 구체적으로, 본 발명은 체내 지속성을 증가시키는 고당화 알파-1 안티트립신 변이체와 인간 성장호르몬이 융합된 지속형 인간 성장호르몬 NexP-hGH 단백질의 특정 이소폼(isoform) 및 이의 제조 방법에 관한 것으로서, 상기 이소폼은 등전점 5.2 이하인, 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 지속형 인간 성장호르몬 NexP-hGH 단백질의 이소폼으로서, 야생형 인간성장호르몬에 비하여 당화가 현저히 증대되어 체내 지속성이 현저히 뛰어날 뿐만 아니라 그 약효 역시 우수한 이점을 지닌다.The present invention relates to a hyperglycemic human growth hormone NexP-hGH protein with enhanced persistence in the body and a method for producing the same. Specifically, the present invention relates to a specific isoform of a sustained human growth hormone NexP-hGH protein fused with a hyperglycosylated alpha-1 antitrypsin mutant and a human growth hormone to increase persistence in the body and a method for producing the same. The isoform is an isoform of a persistent human growth hormone NexP-hGH protein fused with human growth hormone (hGH) and alpha-1 antitrypsin mutant (NexP) having an isoelectric point of 5.2 or less, Not only is remarkably excellent in sustaining properties in the body, but also has an excellent advantage in its drug efficacy.
Description
본 발명은, 고당화되어 체내 지속성이 높은 지속형 인간 성장호르몬 NexP-hGH 단백질 및 이의 제조방법에 관한 것으로, 구체적으로 체내 지속성을 증가시키는 고당화 알파-1 안티트립신 변이체와 인간 성장호르몬이 융합된 지속형 인간 성장호르몬 NexP-hGH 단백질의 특정 이소폼(isoform) 및 이의 제조 방법에 관한 것이다.
The present invention relates to a persistent human growth hormone NexP-hGH protein having high glycosylation and high persistence in the body and a method for producing the same. More specifically, the present invention relates to a NexP-hGH protein having a high glycosylated alpha-1 antitrypsin mutant To a specific isoform of the persistent human growth hormone NexP-hGH protein and a method for producing the same.
일반적으로 천연형 인간 성장호르몬(human growth hormone, hGH)은 191개의 아미노산으로 구성되어 있으며, 약 21,500 달톤의 분자량을 가지는 호르몬이다. 인간 성장호르몬은 뇌하수체 전엽에서 분비되며, 체내에서 뼈, 연골 등의 성장을 촉진시킨다. 이러한 성장호르몬이 결핍되면 저신장증, 심혈관 질환의 위험도 증가, 근육 및 골밀도 감소 등을 유발할 수 있다.In general, human growth hormone (hGH) is composed of 191 amino acids and has a molecular weight of about 21,500 daltons. Human growth hormone is secreted from the anterior pituitary gland and promotes growth of bone, cartilage, etc. in the body. Such a deficiency of growth hormone may lead to short stature, increased risk of cardiovascular disease, decreased muscle and bone density.
성장호르몬 결핍증을 치료하기 위하여 1950년대 후반부터 인간의 뇌하수체에서 유래되거나 또는 유전공학기술로 생산된 성장호르몬이 사용되었으며, 2009년 인간 성장호르몬은 세계적으로 약 3조원의 시장규모를 가지게 되었다. 다만, 성장호르몬의 경우, 매일 피하주사로 투여해야하는 불편함이 있어 환자 편의성의 개선, 증대가 요구되어 왔다. 이러한 요구에 부합하여 지속형 인간 성장호르몬이 개발되고 있으며, 지속성을 높이기 위한 방법으로 서방형 제형, 단백질 또는 당 융합, 당쇄 공학 등이 이용되고 있다. 그 예로, 서방형 제품으로서 제넨텍 사에서 뉴트로핀 디팟(nutropin depot)을 최초로 출시하였으며, 국내에서는 LG 생명과학에서 디클라제를 2007년 출시하였다(대한민국 등록번호 제 10-0236771호 및 제 10-0329336호). 그러나 서방형 치료제는 인체에 원치 않는 면역반응을 야기하고, 굵은 주사기를 사용하여 통증을 유발하며, 낮은 생산수율로 인한 경제성에 문제가 있다. 특히, 뉴트로핀 디팟은 1세대 제품에 비해 유효성이 낮아 시장에서 철수하였다. To treat GHD deficiency, growth hormones derived from human pituitary glands or genetically engineered technology have been used since the late 1950s. In 2009, human growth hormone has reached a market size of about 3 trillion won worldwide. However, in the case of growth hormone, it is required to be administered by subcutaneous injection every day, so that the convenience of patients has been required to be improved and increased. Sustained-release human growth hormone has been developed in accordance with this demand, and a sustained-release formulation, protein or sugar fusion, and sugar chain engineering have been used as methods for improving persistence. For example, Nutropin depot was first released as a slow release product from Genentech, and LG Life Science in Korea released diclase in 2007 (Korean Registration Nos. 10-0236771 and 10-0329336) . However, the sustained-release therapeutic agent causes an unwanted immune response in the human body, causes pain using a thick syringe, and has a problem in economy due to low production yield. In particular, the Neutropin Depot was withdrawn from the market because it was less effective than the first generation.
서방형 제형 외에 단백질의 지속성을 높이기 위한 방법으로서, 고분자 당인 폴리에틸렌글리콜과의 융합(Polyethylene glycol, Sada et al., J.Ferment Bioeng 71, 137-139, 1991), 당쇄 공학(Glycoengineering, 미국등록특허 제 7,217,689호), 다른 단백질과의 융합(국제공개특허 제WO93/15199호)등의 방법을 이용하여 체내의 흡수, 대사, 배설을 시키기도 한다. 다만, 상기 방법들은 낮은 제조 수율로 경제성이 결여되며, 장기간 사용시 면역반응을 야기하고, 결합 과정에서 사용되는 화학 물질이 독성을 야기하는 등 다양한 이유에서 범용적인 반감기 증가방법으로 사용되지 못하고 있다. 따라서, 상기와 같은 단점을 최소화하면서 지속성을 증대시키는 방법을 이용한 지속형 인간 성장호르몬의 개발이 요구되어 왔으며, 대한민국 등록특허 제10-1183262호에서는 체내 단백질인 알파-1 안티 트립신(A1AT)의 변이체와 인간 성장호르몬 결합체 형태의 지속형 인간 성장호르몬 단백질을 개발하여, 단백질의 크기 증가를 통한 반감기의 증대를 꾀한 내용이 개시되어 있다. 이와 더불어, 알파-1 안티트립신에 한 개 이상의 유전자 변이를 통하여 N-당쇄를 추가하여 체내 반감기의 증가를 추가적으로 꾀하였다(대한민국 공개특허 제10-2013-0029713호).
(Sie et al., J. Ferment Bioeng 71, 137-139, 1991), glycoconjugation (Glycoengineering, US Pat. No. 5,304,125), and the like, as a method for increasing the persistence of proteins, 7,217,689), fusion with other proteins (WO93 / 15199), and the like may be used for absorption, metabolism and excretion in the body. However, these methods have not been used as a universal half-life increasing method due to various reasons such as lack of economical efficiency at a low production yield, causing an immune reaction during long-term use, and causing toxicity of chemicals used in the binding process. Therefore, development of a sustained-type human growth hormone by a method of increasing persistence while minimizing the above-mentioned disadvantages has been demanded. In Korean Patent No. 10-1183262, a mutant of alpha-1 antitrypsin (A1AT) And a sustained human growth hormone protein in the form of a human growth hormone conjugate have been developed to increase the half-life by increasing the size of the protein. In addition, N-glycans are added to alpha-1 antitrypsin through one or more gene mutations to further increase the half-life of the body (Korean Patent Publication No. 10-2013-0029713).
체내에 존재하는 단백질은 단백질의 종류에 따라 당화 정도가 반감기 및 유효성에 영향을 미치는 정도가 다르다. 그 예로, 범용적으로 많이 사용되는 단백질 의약품인 에리스로포이에틴과 GCSF(Granulocyte colony-stimulation factor)가 있다. 체내에 존재하는 단백질인 에리스로포이에틴(Erythropoietin, JC Egrie and JK Browne, British J. of Cancer, 84, 3-10, 2001)은 시알산(sialic acid)의 함량에 따라 체내 유효성이 달라진다. 구체적으로, 에리스로포이에틴은 시알산 함량과 체내 유효성 간의 연관성(correlation)이 높으며, 적절한 유효성을 나타내기 위해서는 특정 비율 이상의 시알산이 포함된 고당화 당쇄가 필수적으로 필요하다. 반면, GCSF는 당쇄의 유무에 따라 in vitro 시험에서의 유효성은 25%까지 차이가 나지만, in vivo 유효성에서는 차이가 없는 것으로 알려져 있다(H Bonig et al. Bone marrow transplantation, 28, 259-264, 2001).
Proteins present in the body differ in the degree to which the degree of glycation affects half-life and efficacy, depending on the type of protein. For example, erythropoietin and granulocyte colony stimulation factor (GCSF) are commonly used protein drugs. Erythropoietin (Erythropoietin, JC Egrie and JK Browne, British J. of Cancer, 84, 3-10, 2001), a protein present in the body, differs in vivo effectiveness depending on the content of sialic acid. Specifically, erythropoietin has a high correlation between sialic acid content and body efficacy, and a hyperglycosylated sugar chain containing sialic acid at a specific rate or higher is essential for proper efficacy. In contrast, GCSF is in accordance with the presence or absence of sugar Although the efficacy in vitro tests varies by up to 25%, in vivo It is known that there is no difference in efficacy (H Bonig et al., Bone marrow transplantation, 28, 259-264, 2001).
이에 본 발명자들은 면역원성의 가능성이 낮으면서도 in vitro 및 in vivo 모두에서 높은 지속성 및 약리활성을 보이는 인간 성장 호르몬을 개발하기 위해 예의 노력한 결과, 하나 이상의 아미노산을 변이시켜 특정 부위에 돌연변이가 유발된 알파-1 안티트립신 단백질과 인간 성장호르몬이 융합된 신규한 인간 성장호르몬을 개발하였고, 동물세포에서 제조된 상기 단백질은 in vitro 및 in vivo 모두에서 높은 지속성 및 약리활성을 가짐을 확인하였다. 특히, 본 발명자들은 특정 pI를 가진 NexP-hGH 단백질의 이소폼이 우수한 체내 지속성 및 성장 효과를 지님을 확인하고, 본 발명을 완성하였다.
Therefore, the present inventors have found that although the possibility of immunogenicity is low in vitro and in As a result of intensive efforts to develop human growth hormone exhibiting high persistence and pharmacological activity in both vivo, it has been found that a novel human having a fusion of alpha-1 antitrypsin protein and human growth hormone mutated at a specific site by mutating one or more amino acids Growth hormone was developed, and the protein produced in animal cells was grown in vitro and in vivo . < / RTI > In particular, the present inventors confirmed that the isoform of the NexP-hGH protein having a specific pI had excellent in vivo persistence and growth effect, and completed the present invention.
본 발명의 하나의 목적은 동물세포에서 제조된, 알파-1 안티트립신 변이체(NexP) 및 인간 성장호르몬(hGH)이 융합된 단백질로서, 5.2 이하의 등전점(isoelectric point, pI)을 갖는 지속형 인간 성장호르몬 NexP-hGH 단백질을 제공하는 것이다.One object of the present invention is to provide a fusion protein of alpha-1 antitrypsin mutant (NexP) and human growth hormone (hGH) produced in animal cells, which is a persistent human having an isoelectric point (pI) Growth hormone NexP-hGH protein.
본 발명의 다른 목적은 (a) 알파-1 안티트립신 변이체(NexP) 및 인간 성장호르몬(hGH)이 융합된 지속형 인간 성장호르몬 NexP-hGH 단백질을 포함하는 생물학적 유액을 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피에 적용하는 단계; 및 (b) 등전점(isoelectric point, pI) 차이에 따라 등전점(pI) 5.2 이하인 지속형 성장호르몬 NexP-hGH 단백질을 분리하는 단계를 포함하는, 지속형 인간 성장호르몬 NexP-hGH 단백질의 제조 방법을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition comprising (a) a biological fluid comprising a persistent human growth hormone NexP-hGH protein fused with alpha-1 antitrypsin mutant (NexP) and human growth hormone (hGH) Applying affinity chromatography packed with a resin with a fragment attached thereto; And (b) separating the sustained growth hormone NexP-hGH protein having an isoelectric point (pI) of 5.2 or less according to a difference in isoelectric point (pI). .
본 발명의 또 다른 목적은 상기 방법으로 제조된 지속형 인간 성장호르몬 NexP-hGH 단백질을 제공하는 것이다.
Another object of the present invention is to provide a sustained human growth hormone NexP-hGH protein produced by the above method.
상기의 목적을 달성하기 위한 하나의 양태로서, 본 발명은 동물세포에서 제조된, 알파-1 안티트립신 변이체(NexP) 및 인간 성장호르몬(hGH)이 융합된 단백질로서, 5.2 이하의 등전점(isoelectric point, pI)을 갖는 지속형 인간 성장호르몬 NexP-hGH 단백질을 제공한다.
In one aspect of the present invention, the present invention provides a fusion protein of alpha-1 antitrypsin mutant (NexP) and human growth hormone (hGH) prepared in an animal cell and having an isoelectric point gt; pI) < / RTI > of the human growth hormone NexP-hGH protein.
본 발명에서 용어, "인간 성장호르몬(human growth hormone, hGH)"은 펩타이드 호르몬으로서, 인간의 성장, 세포 생산(cell reproduction) 또는 재생(regeneration)을 가져올 수 있는 호르몬을 의미한다. 상기 인간 성장호르몬은 체내에서 연골 성장판의 세포 분화를 자극하여 성장을 촉진시킬 수 있는 단백질이라면 본 발명에 제한 없이 포함된다. 상기 인간 성장호르몬은 자연적으로 생산된 성장호르몬 및 유전공학기술을 이용하여 생산된 성장호르몬 모두 포함하며, 바람직하게는 유전공학기술을 이용하여 생산된 성장호르몬이나, 이에 제한되지 않는다. 또한, 상기 인간 성장호르몬에 대한 정보는 미국 국립보건원(NCBI) GenBank와 같은 공지의 데이터베이스로부터 얻을 수 있으며, 그 예로 Accession Number가 AAA98618인 인간 성장호르몬 등이 있으나, 이에 제한되지 않는다.
The term "human growth hormone (hGH) " in the present invention means a hormone which can induce human growth, cell reproduction or regeneration as a peptide hormone. The human growth hormone is not limited to the present invention as long as it is a protein capable of promoting growth by stimulating cell differentiation of the cartilage growth plate in the body. The human growth hormone includes naturally produced growth hormone and growth hormone produced using genetic engineering techniques, preferably, but not limited to, a growth hormone produced using genetic engineering techniques. Information on the human growth hormone can be obtained from a known database such as the National Institute of Health (NCBI) GenBank. Examples of such human growth hormone include, but are not limited to, human growth hormone having an accession number of AAA98618.
인간 성장호르몬은 체내에서 연골 성장판의 세포 분화를 자극함으로써 성장을 촉진시킬 수 있으므로, 체내 합성 또는 분비에 있어 문제가 있는 개체에 있어 치료용 단백질로서 중요하게 여겨지고 있다. 다만, 환자의 투여 편의성 및 치료 효과 측면에서 기존 성장호르몬에 비해 반감기가 증대된 지속형 인간 성장호르몬의 개발이 요구되어 왔으며, 이에 본 발명자들은 알파-1 안티트립신 변이체를 개발하고, 이를 인간 성장호르몬에 융합시킨 다음, 동물세포에서 발현시켜 고당화된 인간 성장호르몬 NexP-hGH을 개발하였다. 상기 고당화된 인간성장호르몬 NexP-hGH는 동물세포에서 생산되고 등전점(pI) 5.2 이하인 단백질로서, 등전점 5.2를 초과하는 경우에 비하여 당화 정도가 높아 높은 체내 지속성 및 약효를 나타낸다.
Since human growth hormone can promote growth by stimulating cell differentiation of cartilage growth plate in the body, it is considered to be important as a therapeutic protein in a subject having a problem in synthesis or secretion in the body. However, the development of a sustained-type human growth hormone having an increased half-life compared to the existing growth hormone has been demanded in terms of convenience of administration and therapeutic effect of the patient. Therefore, the present inventors have developed an alpha-1 antitrypsin mutant, And then expressed in animal cells to develop hyperglycosylated human growth hormone NexP-hGH. The hyperglycosylated human growth hormone NexP-hGH is a protein produced in animal cells and having an isoelectric point (pI) of 5.2 or less, which is higher in glycation degree than that in the case of exceeding the isoelectric point of 5.2.
본 발명에서 용어, "지속형 인간 성장호르몬"은 인간 성장호르몬(hGH) 및 알파-1 안티트립신 변이체(NexP)가 융합된 형태의 단백질로서, 본 발명에서 "NexP-hGH"와 혼용되어 사용될 수 있다. 상기 NexP는 본 발명자들에 의해 개발된 체내 지속성을 유지함으로 체내 반감기가 증가된 체내 단백질인 알파-1 안티 트립신(alpha 1-Antitrypsin, A1AT)의 변이체로, 본 발명자들에 의해 명명된 용어이다. 단백질 분해 효소 억제제의 활성을 없앤 알파-1 안티 트립신 변이체 단백질의 서열, 제작 방법 등은 대한민국 등록특허 제10-1183262호 또는 대한민국 공개특허 제10-2013-0029713호에 개시되어 있으며, 대한민국 등록특허 제10-1183262호 명세서 전체 또는 대한민국 공개특허 제10-2013-0029713호 명세서 전체는 본 발명의 참고자료로 포함되나, 이에 제한되지 않는다. In the present invention, the term "persistent human growth hormone" is a protein in which human growth hormone (hGH) and alpha-1 antitrypsin mutant (NexP) are fused and can be used in combination with "NexP-hGH" have. NexP is a variant of alpha 1-antitrypsin (A1AT), which is an in vivo protein having an increased half-life in the body by maintaining in vivo persistence developed by the present inventors, and is a term named by the present inventors. The sequence of the alpha-1 antitrypsin mutant protein, which removes the activity of the protease inhibitor, is disclosed in Korean Patent No. 10-1183262 or Korean Patent Publication No. 10-2013-0029713, 10-1183262 or the entire specification of Korean Patent Publication No. 10-2013-0029713 are included as reference materials of the present invention, but the present invention is not limited thereto.
상기 알파-1 안티트립신은 약 50,000Da의 분자량을 가진, 포유류의 혈액 내에 존재하는 단백질 중의 하나로서, 알파-1 프로테아제 억제제(alpha-1 protease inhibitor)로도 명명된다. 혈액 내에서 추출한 알파-1 안티트립신은 FDA의 허가를 거쳐서 프로라스틴(Prolastin)이라는 상품명으로 폐기종(emphysema) 치료제로 판매되고 있으며, 프로라스틴은 통상적으로 60㎎/㎏의 용량으로 1주 간격으로 정맥 주사로 인체에 투여되며, 인체에서의 안전성이 입증이 된 단백질이다. 또한, 알파-1 안티 트립신의 프로테아제 억제제로의 역할 및 구조 등은 이미 잘 알려져 있다(Elliott, P. et al., JMB 275, 419-425, 1998). 또한, 상기 알파-1 안티트립신은 자연계에 100여 종 이상의 대립유전자(allele)가 존재하고, 표현형은 IEF(isoelectric focusing) 유형에 따라 A에서 Z로 구분한다(Stoller et al., The Lancet, 365, 2225-2236, 2005). 이중 가장 많은 M 대립 유전자는 정상형으로 아미노산 서열 변이에 의해 다시 M1(Val213), M2, M3와 같이 여러 가지 아형(subtype)으로 구분된다. 따라서, 본 발명에 사용된 알파-1 안티트립신은 자연계에 존재하는 특정 아형이며, 다른 아형에 대하여도 동일한 효과를 얻을 수 있다. 상기 알파-1 안티트립신 단백질에 대한 정보는 미국 국립보건원(NCBI) GenBank와 같은 공지의 데이터베이스로부터 얻을 수 있으며, 그 예로 Accession Number가 AAH11991인 야생형 알파-1 안티트립신 단백질일 수 있으나, 이에 제한되지 않는다. 상기 야생형 알파-1 안티트립신 단백질의 서열을 서열번호 1에 나타내었다. The alpha-1 antitrypsin is also one of the proteins present in the blood of mammals, having a molecular weight of about 50,000 Da, also referred to as alpha-1 protease inhibitor. Alpha-1 antitrypsin extracted from the blood is sold under the FDA's approval as Prolastin as an emphysema treatment, and prolactin is usually administered at a dose of 60 mg / Is administered to the human body by intravenous injection, and has been proven to be safe in human body. In addition, the role and structure of alpha-1 antitrypsin as a protease inhibitor is well known (Elliott, P. et al., JMB 275, 419-425, 1998). The above-mentioned alpha-1 antitrypsin has more than 100 kinds of alleles in nature, and the phenotype is divided into A to Z according to IEF (isoelectric focusing) type (Stoller et al., The Lancet, 365 , 2225-2236, 2005). The most common M allele is a normal type and is divided into several subtypes such as M1 (Val 213 ), M2 and M3 by amino acid sequence mutation. Therefore, the alpha-1 antitrypsin used in the present invention is a specific subtype existing in nature, and the same effect can be obtained for other subtypes. Information on the alpha-1 antitrypsin protein can be obtained from a well-known database such as the National Institute of Health (NCBI) GenBank, including but not limited to the wild type alpha-1 antitrypsin protein with Accession Number AAH 11991 . The sequence of the wild-type alpha-1 antitrypsin protein is shown in SEQ ID NO: 1.
이러한 알파-1 안티트립신은 특정부위 돌연변이 유발(site-directed mutagenesis) 방법을 이용하여 하나 이상의 아미노산 잔기를 변형시켜 고유한 체내 활성을 없애고 반감기를 증가시킬 수 있다. 상기 아미노산 잔기의 변형을 통하여 N-당화 부위를 생성하여 알파-1 안티트립신의 프로테아제 억제제의 활성을 중화시키는 동시에 체내 주입시에 아미노산 치환에 의한 면역원성 가능성을 최소화할 수 있으며, 유리 시스테인기에 의한 이중체 형성 등을 제거할 수 있다. 이때 하나 이상의 아미노산의 변이는 P2 위치인 서열번호 1의 알파-1 안티트립신 단백질의 357번째 아미노산인 프롤린(Proline, P)을 변이시킨 것을 특징으로 할 수 있으며, 보다 구체적으로 이를 아스파라진(Asparagine, N)으로 변이시킨 것을 특징으로 할 수 있다. 또한, 알파-1 안티트립신 변이체는 서열번호 1의 알파-1 안티트립신 단백질에서 9번째 아미노산인 글루타민(Glutamine, Q)의 아스파라진으로의 변이, 232번째 아미노산인 시스테인(Cysteine, C)의 세린(Serine, S)으로의 변이 또는 359번째 세린(Serine, S)의 트레오닌(Threonine, T)으로의 변이를 포함할 수 있으며, 이때 P2 위치인 357번째 아미노산인 프롤린이 아스파라진으로 변이되고, 상기 변이 중 하나 이상을 추가로 포함할 수 있으나, 이에 제한되지 않는다.Such alpha-1 antitrypsin may be modified by one or more amino acid residues using a site-directed mutagenesis method to eliminate intrinsic activity and increase half-life. By modifying the amino acid residues, an N-glycosylation site is generated to neutralize the activity of a protease inhibitor of alpha-1-antitrypsin. In addition, the possibility of immunogenicity due to amino acid substitution during injection into the body can be minimized. Sieve formation and the like can be removed. In this case, the mutation of one or more amino acids may be characterized by mutation of Proline (P), which is the 357th amino acid of the alpha-1 antitrypsin protein of SEQ ID NO: 1 which is at the P2 position. More specifically, it may be asparagine N). ≪ / RTI > Also, the alpha-1 antitrypsin mutant has a mutation of the 9th amino acid Glutamine (Q) to asparagine in the alpha-1 antitrypsin protein of SEQ ID NO: 1, a serine of cysteine (C) Serine, S) or the 359th serine (S) to threonine (T), wherein proline, the 357th amino acid at position P2, is mutated to asparagine, But it is not limited thereto.
상기 지속형 인간 성장호르몬 NexP-hGH은 이러한 알파-1 안티 트립신(A1AT)의 변이체를 인간 성장호르몬의 N-말단 또는 C-말단에 유전자 재조합 방식으로 융합한 단백질이다. 특히, 상기 지속형 인간 성장호르몬 NexP-hGH은 인간 성장호르몬의 N-말단에, 9번째 아미노산인 글루타민 및 357번째 아미노산인 프롤린이 모두 아스파라진으로 변이된 알파-1 안티트립신 단백질이 융합된 단백질(hGH-A1AT(Q9N, P357N))일 수 있으나, 이에 제한되지는 않는다. The persistent human growth hormone NexP-hGH is a fusion protein of a mutant of alpha-1 antitrypsin (A1AT) to the N-terminal or C-terminal of human growth hormone by gene recombination. In particular, the persistent human growth hormone NexP-hGH is a fusion protein of alpha-1 antitrypsin protein in which the 9th amino acid glutamine and the 357th amino acid proline are all substituted with asparagine at the N-terminus of human growth hormone hGH-A1AT (Q9N, P357N)).
본 발명에서 용어, "고당화된 지속형 인간 성장호르몬"은 알파-1 안티트립신 변이체와 인간 성장호르몬을 포함하는 형태의 단백질로서, 그 당화 정도가 자연상태에서 발견되는 야생형 단백질보다 당화 정도가 높고 등전점 값이 5.2 이하로 당화된 지속형 인간 성장호르몬을 의미한다.As used herein, the term "hyperglycosylated sustained human growth hormone" refers to a protein in the form of an alpha-1 antitrypsin mutant and a human growth hormone, the glycation degree of which is higher than that of a wild- Means persistent human growth hormone glycated to an isoelectric point value of less than 5.2.
본 발명에서는, 상기 지속형 인간성장호르몬의 당화 정도가 높아질수록 높은 체내 지속성을 가질 수 있음을 확인하였으며, 그 중에서도 지속형 인간 성장호르몬의 등전점이 5.2 이하일 때 in vitro 및 in vivo 모두에서 현저하게 높은 체내 지속성 및 약물 활성을 나타냄을 규명하였다.In the present invention, it was confirmed that the glycation degree of the long-acting human growth hormone The increase can have a high vivo persistence, when the isoelectric point of the long-acting human growth hormone is less than 5.2, most of all in vitro and in vivo, respectively. < tb >< TABLE >
상기 고당화된 지속형 인간 성장호르몬은 등전점이 5.2 이하, 바람직하게는3.5 내지 5.2이나, 이에 제한되지 않는다.The hyperglycosylated sustained-type human growth hormone has an isoelectric point of 5.2 or less, preferably 3.5 to 5.2, but is not limited thereto.
여기서, 상기 당화 부위는 탄수화물 구조(carbohydrate structure)의 부착과 같은 글리코실화가 일어날 수 있는 폴리펩타이드 내의 아미노산 잔기 또는 부위로서, 이러한 부위로는 N-글리코실화 부위 또는 O-글리코실화 부위 등이 대표적이다. 보존적인 N-글리코실화 부위로는 Asn-X-Ser 또는 Asn-X-Thr이 있으며, 여기서 X는 임의의 아미노산이나, 이에 제한되지 않는다.
Herein, the glycation site is an amino acid residue or a site in a polypeptide in which glycosylation such as adhesion of a carbohydrate structure can take place. Examples of such sites are an N-glycosylation site or an O-glycosylation site . Conservative N-glycosylation sites include Asn-X-Ser or Asn-X-Thr, where X is any amino acid, but is not limited thereto.
상기 고당화된 지속형 인간 성장호르몬은 상기 지속형 인간 성장호르몬을 코딩하는 폴리뉴클레오티드를 포함하는 발현 벡터가 도입된 동물세포를 배양하여 생산할 수 있다. The hyperglycosylated sustained human growth hormone may be produced by culturing animal cells into which an expression vector containing the polynucleotide encoding the persistent human growth hormone has been introduced.
본 발명에서 용어, "동물세포"는 본 발명의 지속형 인간 성장호르몬을 발현시킬 수 있는 세포로서, 당화를 가져오는 세포라면 그 종류가 특별히 제한되지 않으나, 그 예로 CHO 세포, BHK 세포, Vero 세포, HeLa 세포, MDCK 세포, 293 세포 및 3T3 세포가 있으나, 이에 제한되지 않는다. As used herein, the term "animal cell" refers to a cell capable of expressing the persistent human growth hormone of the present invention. The type of the cell is not particularly limited as long as it is a cell that induces glycation. Examples thereof include CHO cells, BHK cells, Vero cells , HeLa cells, MDCK cells, 293 cells, and 3T3 cells.
본 발명의 일 실시예에서는 9번 및 357번째 아미노산이 모두 아스파라진으로 치환된 알파-1 안티트립신이 융합된 인간 성장호르몬 단백질을 제조하였으며, 상기 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 CHO 세포에 도입하여, Stable 세포주를 제작하였다(실시예 1). 또한, 음이온 교환 수지 크로마토그래피, 소수성 크로마토그래피 및 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피를 순차적으로 수행하고 등전점 차이에 따라 NexP-hGH 단백질을 수득한 결과, 이소폼 1 내지 3 중 낮은 pI를 가지는 이소폼 1이 이소폼 2 및 3에 비하여 높은 당화를 가지며, 이소폼 3에 비하여 낮은 pI를 가지는 이소폼 2가 이소폼 3에 비하여 높은 당화를 나타냄을 확인하였다(도 1 및 2). 또한, 인간 성장호르몬 약역학을 확인한 결과, 당화 정도가 높아질수록 본 발명의 지속형 인간 성장호르몬의 약역학이 뛰어났으며, 특히 양성대조군과 대등하거나 우수한 결과를 보였다(도 3). 또한 이소폼 1 및 이소폼 2는 양성대조군 대비 시험동물의 몸길이를 현저히 늘려주는 것을 확인하였다(도 4). 즉, 본 발명의 고당화된 지속형 인간 성장호르몬은 양성대조군에 비하여 현저히 높은 유효성을 보이는 결과를 나타내었다.
In one embodiment of the present invention, a human growth hormone protein fused with alpha-1 antitrypsin in which all of the 9th and 357th amino acids were substituted with asparagine was prepared and a vector containing the polynucleotide encoding the protein was inserted into CHO cells To prepare a Stable cell line (Example 1). In addition, affinity chromatography packed with an anion exchange resin chromatography, hydrophobic chromatography, and a resin having an anti-alpha-1 antitrypsin antibody fragment attached thereto was sequentially performed and NexP-hGH protein was obtained according to isoelectric point difference. As a result, It has been confirmed that
또 다른 양태로서, 본 발명은 (a) 알파-1 안티트립신 변이체(NexP) 및 인간 성장호르몬(hGH)이 융합된 지속형 인간 성장호르몬 NexP-hGH 단백질을 포함하는 생물학적 유액을 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피에 적용하는 단계; 및 (b) 등전점(isoelectric point, pI) 차이에 따라 등전점(pI) 5.2 이하인 지속형 성장호르몬 NexP-hGH 단백질을 분리하는 단계를 포함하는, 지속형 인간 성장호르몬 NexP-hGH 단백질의 제조 방법을 제공한다.
In another aspect, the present invention provides a method for the treatment and / or prophylaxis of (a) biological latex comprising a sustained human growth hormone NexP-hGH protein fused with alpha-1 antitrypsin mutant (NexP) and human growth hormone To an affinity chromatography packed with a resin to which a trypsin antibody fragment is attached; And (b) separating the sustained growth hormone NexP-hGH protein having an isoelectric point (pI) of 5.2 or less according to a difference in isoelectric point (pI). do.
상기 알파-1 안티트립신 변이체, 인간 성장호르몬 및 지속형 인간 성장호르몬에 대해서는 상기에서 설명한 바와 같다.
The above-mentioned alpha-1 antitrypsin mutant, human growth hormone and sustained human growth hormone are as described above.
상기 제조 방법의 각 단계를 구체적으로 설명하면 다음과 같다.
Each step of the above manufacturing method will be described in detail as follows.
상기 (a) 단계는 지속형 인간 성장호르몬인 NexP-hGH를 포함하는 생물학적 유액을 안티 알파-1 안티트립신 항체 단편이 부착된 수지로 충진된 친화성 크로마토그래피에 적용하는 단계이다. The step (a) is a step of applying a biological fluid containing NexP-hGH, which is a sustained human growth hormone, to an affinity chromatography packed with a resin having an anti-alpha-1 antitrypsin antibody fragment attached thereto.
상기 단계는 NexP와 항체 단편 간의 친화도를 통하여 NexP-hGH를 컬럼에 부착시키기 위한 단계이다.This step is for attaching NexP-hGH to the column through the affinity between NexP and the antibody fragment.
본 발명에서 용어, "지속형 인간 성장호르몬인 NexP-hGH를 포함하는 생물학적 유액"은 지속형 인간 성장호르몬 NexP-hGH 단백질을 생산하는 세포의 배양 상등액(cell culture supernant), 상기 세포의 파쇄물(cell extract), 또는 이의 부분 정제된 형태일 수 있으나, 이에 제한되지 않는다. 특히, 상기 생물학적 유액은 바람직하게는 상기 지속형 인간 성장호르몬을 코딩하는 폴리뉴클레오티드를 포함하는 발현 벡터가 도입된 동물세포를 배양하여 수득한 배양 상등액 또는 상기 세포의 파쇄물이나, 이에 제한되지 않는다.The term "biological fluid containing NexP-hGH, which is a persistent human growth hormone" in the present invention refers to a cell culture supernant of a cell producing a sustained human growth hormone NexP-hGH protein, extract, or a partially purified form thereof. In particular, the biological fluid is preferably a culture supernatant obtained by culturing an animal cell into which an expression vector containing a polynucleotide encoding the persistent human growth hormone is introduced, or a disruption of the cell, but is not limited thereto.
본 발명에서 용어, "부분 정제(partially purified)"는 크로마토그래피와 같은 분류 과정(fractionation procedure)을 하나 이상 수행하였으나 목적하는 pI값을 가지는 NexP-hGH 단백질 외에도 다른 단백질이 존재하는 상태를 의미한다. 상기 부분 정제 과정은 특별히 그 종류가 제한되지 않으며, 그 예로 소수성 크로마토그래피 또는 음이온 교환 수지 크로마토그래피일 수 있으나, 이에 제한되지 않는다. 상기 부분 정제는 소수성 크로마토그래피 및 음이온 교환 수지 크로마토그래피로 이루어진 군으로부터 선택되는 하나 이상의 방법을 이용하여 정제할 수 있으며, 바람직하게는 음이온 교환 수지 크로마토그래피 또는 소수성 수지 크로마토그래피에 지속형 인간 성장호르몬 NexP-hGH 단백질을 생산하는 세포의 배양 상등액 또는/및 상기 세포의 파쇄물을 적용하여 용출액을 생성시키거나, 보다 바람직하게는 음이온 교환 수지 크로마토그래피 및 소수성 수지 크로마토그래피를 순차적으로 적용하여 용출액을 생성시키는 것일 수 있으나, 이에 제한되지 않는다.
The term "partially purified" in the present invention means a state in which at least one fractionation procedure such as chromatography is performed but other proteins are present in addition to the NexP-hGH protein having the desired pI value. The partial purification process is not particularly limited, and examples thereof include, but are not limited to, hydrophobic chromatography or anion exchange resin chromatography. The partial purification may be carried out using one or more methods selected from the group consisting of hydrophobic chromatography and anion exchange resin chromatography, preferably by anion exchange resin chromatography or hydrophobic resin chromatography using a sustained-type human growth hormone NexP to produce an eluate by applying a culture supernatant of a cell producing the -hGH protein or a lysate of the cell or, more preferably, anion exchange resin chromatography and hydrophobic resin chromatography sequentially to produce an eluate But is not limited thereto.
상기 (b) 단계는 pI 차이에 따라 등전점(pI) 5.2 이하인 지속형 성장호르몬을 분리하는 단계이다. The step (b) separates the persistent growth hormone having an isoelectric point (pI) of 5.2 or less according to the difference in pI.
상기 항체 단편이 부착된 수지로부터 분리하는 단계에서, 지속형 인간 성장호르몬인 NexP-hGH의 당사슬 패턴 및 등전점이 다른 구조적 아형(isoform)은 MgCl2 농도에 따라 분리용출이 가능하다. In the step of separating from the resin to which the antibody fragment is attached, the structural isoform of NexP-hGH, which is a persistent human growth hormone, having different sugar chain pattern and isoelectric point can be separated and eluted according to MgCl 2 concentration.
따라서, 상기 (b) 단계는 등전점(pI) 5.2 이하인 지속형 성장호르몬을 분리용출하기 위하여, 0 내지 1000mM MgCl2가 포함된 트리스 완충용액, 바람직하게는 0 내지 300mM MgCl2, 더욱 바람직하게는 0 내지 200mM MgCl2 , 더욱더 바람직하게는 200mM MgCl2가 포함된 트리스 완충용액을 적용하여 등전점(pI) 5.2 이하인 지속형 성장호르몬을 분리용출할 수 있으나, 이에 제한되지 않는다.Accordingly, the step (b) isoelectric point (pI) to elute separate the long-acting growth hormone less than 5.2, a Tris buffer solution containing 0 to 1000mM MgCl 2, preferably 0 to 300mM MgCl 2, more preferably 0 to 200mM MgCl 2, and still more preferably by applying a tris buffer solution containing 200mM MgCl 2 isoelectric point (pI), but to separate the long-acting GH, eluting 5.2 or less, but is not limited thereto.
분리용출을 위한 트리스 완충용액에 있어서, MgCl2은 선택적으로 첨가하거나 첨가하지 않을 수 있다.
In the Tris buffer solution for the separation elution, MgCl 2 may be optionally added or not added.
또 다른 양태로서, 본 발명은 상기 방법으로 제조된 지속형 인간 성장호르몬 NexP-hGH 단백질을 제공한다.In another aspect, the present invention provides a sustained human growth hormone NexP-hGH protein produced by the above method.
상기 방법 및 지속형 인간 성장호르몬 NexP-hGH 단백질에 대해서는 상기에서 설명한 바와 같다.
The above method and the persistent human growth hormone NexP-hGH protein are as described above.
본 발명의 고당화 지속형 인간성장호르몬 및 이의 제조방법은 기존의 인간성장호르몬에 비하여 당화가 현저히 증대되어, 시판되는 디클라제와 같은 인간성장호르몬에 비하여 지속성이 현저히 뛰어날 뿐만 아니라 그 약효 역시 우수하므로, 인간 성장호르몬이 필요한 분야에 유용하게 사용될 수 있다.
The hyperglycemia sustained-type human growth hormone and the method for producing the same of the present invention remarkably increase glycosylation as compared with the existing human growth hormone, and are remarkably excellent in persistency and also excellent in drug efficacy as compared with human growth hormone such as commercially available diclase, And can be usefully used in fields requiring human growth hormone.
도 1 및 2는, pI의 차이에 따른 지속형 인간 성장호르몬의 분획을 나타낸 것이다.
도 3은, 본 발명의 고당화 지속형 인간 성장호르몬 이소폼 1 내지 3의 몸무게에 미치는 영향을 확인한 도이다.
도 4는, 본 발명의 고당화 지속형 인간 성장호르몬 이소폼 1 내지 3의 몸길이를 측정한 결과를 나타낸 도이다.Figures 1 and 2 show the fraction of persistent human growth hormone according to the difference in pI.
FIG. 3 is a chart for confirming the effect of the hyperglycemia sustained-type human
FIG. 4 is a graph showing the results of measurement of body length of the hyperglycemia-sustained human
이하, 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. 이 분야에서 통상의 지식을 가진 자는 주어진 상황에 따라 통상적으로 사용되는 벡터 및 배양조건 등을 적절히 선택할 수 있다.
Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples. Those skilled in the art can appropriately select vectors and culturing conditions conventionally used according to a given situation.
실시예Example : 인간성장호르몬/알파-1 : Human Growth Hormone / Alpha-1 안티트립신Antitrypsin 융합체의Fused 제조 및 Manufacturing and 융합체Fusant 발현 세포주의 제조 Preparation of Expression Cell Lines
pSGHV0(GenBank Accession No. AF285183)에서 shGH, His tag, TEV site를 제거한 개량벡터 pAV1을 이용하여 인간성장호르몬/알파-1 안티트립신 융합체를 클로닝하였으며, 단백질 고유의 신호 서열(signal sequence)을 이용하여 세포 외로 분비되도록 하였다.The human growth hormone / alpha-1 antitrypsin fusions were cloned using the improved vector pAV1 from which shGH, His tag and TEV site were removed from pSGHV0 (GenBank Accession No. AF285183), and using the protein specific signal sequence And secreted out of the cell.
또한 상기 융합체를 지속적으로 발현하는 세포주(stable cell line) 구축을 위한 선별마커로 DHFR 시스템을 도입하였으며, 이를 위하여 IRES-DHFR 유전자를 pAV1 벡터에 삽입하였다. 발현량 증대를 위해 신호 서열에 Kozac 서열을 추가로 삽입하였다. 표적 단백질인 hGH-A1AT(Q9N, P357N)은 A1AT의 N 말단에 hGH를 융합하는 방식으로 도입하였다.In addition, the DHFR system was introduced as a selection marker for constructing a stable cell line that continuously expresses the fusion construct. To this end, the IRES-DHFR gene was inserted into the pAV1 vector. Kozac sequence was further inserted into the signal sequence to increase the expression level. The target protein hGH-A1AT (Q9N, P357N) was introduced by fusing hGH to the N-terminus of A1AT.
그 다음, 이렇게 제조된 클론을 CHO DG44 세포주에 유전자 도입을 하여, MTX(methotrexate) 50nM에서 시작하여 2배수로 늘려주며 4μM까지 선별을 진행하여, Stable 세포주를 확보하였다. Then, the clone thus prepared was transfected into CHO DG44 cell line and MTX (methotrexate) was started at 50 nM and doubled to 4 μM to obtain a stable cell line.
다만, 이 분야에서 통상의 지식을 가진 자는 주어진 상황에 따라 통상적으로 사용되는 벡터 및 세포주를 적절히 선택하여 적용할 수 있다.
However, those of ordinary skill in the art can appropriately select and apply vectors and cell lines that are conventionally used according to a given situation.
실험예Experimental Example 1: 지속형 인간 성장호르몬/알파-1 1: Persistent human growth hormone / alpha-1 안티트립신Antitrypsin 융합체Fusant 발현 세포주의 배양 및 Cultivation of the expressing cell line and 융합체Fusant 생산 production
상기에서 얻어진 세포주에서 고당화된 성장호르몬/알파-1 안티트립신 융합체를 고수율로 얻기 위하여, 본 발명의 지속형 성장호르몬인 성장호르몬/알파-1 안티트립신 융합체을 통상적인 세포배양 조건에서 발현시켰다. 다만, 이 분야에서 통상의 지식을 가진 자는 주어진 상황에 따라 통상적으로 사용되는 배양조건 등을 적절히 선택하여 적용할 수 있다.
In order to obtain a hyperglycosylated growth hormone / alpha-1 antitrypsin fusion product from the cell line obtained above in high yield, the growth hormone / alpha-1 antitrypsin fusion, the sustained growth hormone of the present invention, was expressed under usual cell culture conditions. However, a person having ordinary skill in the art can appropriately select and apply culture conditions normally used according to a given situation.
실험예Experimental Example 2: 인간 성장호르몬/알파-1 2: human growth hormone / alpha-1 안티트립신Antitrypsin 융합체의Fused pIpI 별 분획Star fraction
상기 실시예에서 얻어진 발현 세포주를 현탁배양하여 얻어진 세포배양액으로 분비된 본 발명의 신규한 지속형 성장호르몬을 정제하였다. The novel sustained growth hormone of the present invention secreted into a cell culture obtained by suspension culturing of the expression cell line obtained in the above Example was purified.
구체적으로, 배양액을 여과하여 세포를 제거한 후 상등액만 취하고, 평형 완충용액(20mM 소듐 포스페이트, pH 8.0)을 이용하여 한외 여과 시스템(분자량 컷 오프 30,000)을 이용하여 정용여과하였다. 그 다음, 이를 Q-세파로즈(Q-sepharose, GE Healthcare, 미국) 컬럼에 주입하고, 평형 완충용액과 50mM NaCl이 포함된 20mM 소듐 포스페이트 완충액(pH 8.0)으로 불순 단백질들을 제거한 다음, 190mM NaCl이 포함된 20mM 소듐 포스페이트 완충용액(pH 8.0)을 흘려, 지속형 성장호르몬이 포함된 용액을 회수하였다.Specifically, the culture solution was filtered to remove the supernatant, and the supernatant was collected and subjected to dental filtration using an ultrafiltration system (molecular weight cut-off of 30,000) using an equilibration buffer solution (20 mM sodium phosphate, pH 8.0). Then, this was injected into a Q-sepharose (GE Healthcare, USA) column, and impurity proteins were removed with 20 mM sodium phosphate buffer (pH 8.0) containing 50 mM NaCl in equilibrium buffer. Then, 190 mM NaCl The solution containing the sustained growth hormone was recovered by flowing 20 mM sodium phosphate buffer (pH 8.0) contained therein.
Q-세파로즈 컬럼 용출액에 4M NaCl/20mM 소듐 포스페이트 완충액(pH 7.5)을 첨가하여 약 2.5M NaCl/20mM 소듐 포스페이트(pH 7.5)가 되도록 하여 페닐-세파로즈(GE Healthcare, 미국) 로딩액을 준비하였다. 그 다음, 이를 페닐-세파로즈 컬럼에 로딩하고, 2.5M NaCl이 포함된 20mM 소듐 포스페이트 완충액으로 컬럼을 세척하였다. 그 다음, 20mM 소듐 포스페이트 완충액(pH 7.5)을 이용하여 지속형 인간 성장호르몬이 포함된 용액을 회수하였다.Sepharose (GE Healthcare, USA) loading solution was prepared by adding 4M NaCl / 20mM sodium phosphate buffer (pH 7.5) to the Q-sepharose column eluate to make about 2.5M NaCl / 20mM sodium phosphate Respectively. It was then loaded onto a phenyl-sepharose column and the column was washed with 20 mM sodium phosphate buffer containing 2.5 M NaCl. The solution containing the persistent human growth hormone was then recovered using 20 mM sodium phosphate buffer (pH 7.5).
페닐 세파로즈 컬럼 용출액을 150mM NaCl이 포함된 트리스(Tris) 완충용액(pH 7.4)을 이용하여 한외 여과 시스템(분자량 컷 오프 30,000)을 이용하여 정용여과하였다. 그 다음, 이를 안티 알파1-안티트립신(A1AT) 항체 단편이 부착된 수지(GE Healthcare, 이하 'A1AT'로 명명)에 로딩하여 컬럼에 결합시킨 후, 50mM MgCl2가 포함된 트리스(Tris) 완충용액(pH 7.4)으로 세척하였다. 이후, 100mM MgCl2이 포함된 트리스(Tris) 완충용액(pH 7.4), 200mM MgCl2이 포함된 트리스(Tris) 완충용액(pH 7.4), 300mM MgCl2이 포함된 트리스(Tris) 완충용액(pH 7.4)을 순차적으로 흘려 pI 차이에 따른 본 발명의 지속형 성장호르몬을 분획하여 용출하였다. The phenylsaparose column eluate was filtered by diafiltration using an ultrafiltration system (molecular weight cut-off 30,000) using a Tris buffer solution (pH 7.4) containing 150 mM NaCl. Then, this anti-alpha-1-antitrypsin (A1AT), the antibody fragment is attached to a resin loaded in a (GE Healthcare, with less 'A1AT' Unnamed) was coupled to the column, 50mM MgCl 2 a Tris (Tris) buffer containing the Solution (pH 7.4). Thereafter, a Tris buffer solution (pH 7.4) containing 100 mM MgCl 2 , a Tris buffer solution (pH 7.4) containing 200 mM MgCl 2 , a Tris buffer solution containing 300 mM MgCl 2 (pH 7.4) were sequentially flowed to separate and elute the sustained growth hormone of the present invention according to the pI difference.
이때, 100mM MgCl2/트리스 완충용액(pH 7.4)으로 용출된 본 발명의 지속형 성장호르몬을 이소폼(isoform) 1로, 200mM MgCl2/트리스 완충용액(pH 7.4)으로 용출된 본 발명의 지속형 성장호르몬을 이소폼(isoform) 2로, 300mM MgCl2/트리스 완충용액(pH 7.4)으로 용출된 본 발명의 지속형 성장호르몬을 이소폼(isoform) 3으로, 각각 명명하였다(도 1 및 2). At this time, the sustained growth hormone of the present invention, which was eluted with 100 mM MgCl 2 / Tris buffer solution (pH 7.4), was dissolved in 200 mM MgCl 2 / Tris buffer solution (pH 7.4) in
상기 방법 외에도 이 분야에서 통상의 지식을 가진 자는 등전점에 따라 단백질을 분리하기 위한 통상의 방법을 적절히 선택하여 적용할 수 있다.
In addition to the above methods, those skilled in the art can appropriately select and apply a conventional method for separating proteins according to the isoelectric point.
실험예Experimental Example 3: 인간 성장호르몬/알파-1 3: human growth hormone / alpha-1 안티트립신Antitrypsin 융합체의Fused 약역학Pharmacokinetics 실험 Experiment
본 발명에 따른 지속형 성장호르몬 분획의 약역학을 확인하기 위하여 하기와 같은 실험을 수행하였다.
The following experiment was conducted to confirm the pharmacokinetics of the sustained growth hormone fraction according to the present invention.
뇌하수체 제거 랫트에 상기 이소폼 1 내지 3을 0.6mg/kg의 농도로 피하(S.C.) 투여하여 14일 차까지 몸무게의 변화 및 몸길이를 측정하였고, 그 결과를 각각 도 3 및 4에 나타내었다. The isoforms 1-3 were administered subcutaneously (SC) at a concentration of 0.6 mg / kg to the rat pituitary-removing rats, and body weight and body weight were measured until
그 결과, 도 3에 나타난 바와 같이, 음성대조군인 PBS 투여군에서는 무게변화가 없었으나. 이소폼 1 내지 3 및 양성대조군인 유트로핀(Eutropin) 및 디클라제(Declage) 투여 군에서는 현저한 무게 증가가 나타났다. 특히, 이소폼 1 및 2는 양성대조군보다 무게 증가율이 10일 차에 더 컸으며, 이소폼 3은 양성대조군보다 다소 저조한 증가율을 보였다. As a result, as shown in Fig. 3, there was no change in weight in the PBS control group as a negative control group. Significant weight gain was observed in the isoforms 1-3 and in the positive control groups Eutropin and Declage. Especially,
또한, 몸길이 측정 결과의 경우, 이소폼 1 및 2는 양성대조군인 디클라제보다 몸길이 증가율이 컸으나, 이소폼 3은 다소 적은 결과를 나타내었다(도 4).
In addition, in the case of body weight measurement,
상기와 같은 결과는 같은 종류의 지속형 성장 호르몬이라 하더라도, 당쇄의 정도에 따라 효능의 정도에 차이가 있음을 나타내는 것으로, 특히 본 발명의 방법으로 제조된, 5.2 이하의 pI를 가진 NexP-hGH 단백질이 고당화 단백질로서, 체내 지속성 및 높은 성장효과를 지님을 시사하는 것이다.
The above results indicate that even though the same type of sustained growth hormone is different in the degree of the effect according to the degree of sugar chains, it is possible to obtain a NexP-hGH protein having a pI of 5.2 or less, prepared by the method of the present invention Suggesting that it is a hyperglycosylated protein and has persistence in the body and a high growth effect.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시 예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.
From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.
<110> CJ CheilJedang Corporation Alteogen, Inc <120> Hyperglycosylated long-acting human growth hormone and method for preparing the same <130> PA120923KR <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 394 <212> PRT <213> Homo sapiens <400> 1 Glu Asp Pro Gln Gly Asp Ala Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Ile Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390 <110> CJ CheilJedang Corporation Alteogen, Inc <120> Hyperglycosylated long-acting human growth hormone and method for preparing the same <130> PA120923KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 394 <212> PRT <213> Homo sapiens <400> 1 Glu Asp Pro Gln Gly Asp Ala Gln Lys Thr Asp Thr Ser His His 1 5 10 15 Asp Gln Asp His Pro Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu 20 25 30 Phe Ala Phe Ser Leu Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr 35 40 45 Asn Ile Phe Phe Ser Ser Val Ser Ile Ala Thr Ala Phe Ala Met Leu 50 55 60 Ser Leu Gly Thr Lys Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu 65 70 75 80 Asn Phe Asn Leu Thr Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe 85 90 95 Gln Glu Leu Leu Arg Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu 100 105 110 Thr Thr Gly Asn Gly Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp 115 120 125 Lys Phe Leu Glu Asp Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr 130 135 140 Val Asn Phe Gly Asp Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr 145 150 155 160 Val Glu Lys Gly Thr Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu 165 170 175 Asp Arg Asp Thr Val Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly 180 185 190 Lys Trp Glu Arg Pro Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe 195 200 205 His Val Asp Gln Val Thr Thr Val Lys Val Pro Met Met Lys Arg Leu 210 215 220 Gly Met Phe Asn Ile Gln His Cys Lys Lys Leu Ser Ser Trp Val Leu 225 230 235 240 Leu Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile Phe Leu Pro Asp 245 250 255 Glu Gly Lys Leu Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile 260 265 270 Thr Lys Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu 275 280 285 Pro Lys Leu Ser Ile Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly 290 295 300 Gln Leu Gly Ile Thr Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly 305 310 315 320 Val Thr Glu Glu Ala Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala 325 330 335 Val Leu Thr Ile Asp Glu Lys Gly Thr Glu Ala Ala Gly Ala Met Phe 340 345 350 Leu Glu Ala Ile Pro Met Ser Pro Pro Glu Val Lys Phe Asn Lys 355 360 365 Pro Phe Val Phe Leu Met Ile Asp Gln Asn Thr Lys Ser Pro Leu Phe 370 375 380 Met Gly Lys Val Val Asn Pro Thr Gln Lys 385 390
Claims (8)
A fusion protein of alpha-1 antitrypsin mutant (NexP) and human growth hormone (hGH) produced in animal cells and having a constant human growth hormone NexP-hGH protein having an isoelectric point (pI) of 5.2 or less , The alpha-1 antitrypsin mutant (NexP) is a serine of cysteine which is the 232th amino acid, a mutation of the 9th glutamine of the wild type alpha-1 antitrypsin protein to asparagine The mutation of proline to asparagine, the 357th amino acid, and the mutation of serine to threonine, which is the 359th amino acid. , A sustained human growth hormone NexP-hGH protein.
(b) 등전점(isoelectric point, pI) 차이에 따라 등전점(pI) 5.2 이하인 지속형 성장호르몬 NexP-hGH 단백질을 분리하는 단계를 포함하는, 지속형 인간 성장호르몬 NexP-hGH 단백질의 제조 방법으로서, 상기 알파-1 안티트립신 변이체(NexP)는 야생형 알파-1 안티트립신 단백질의 9번째 글루타민(glutamine)의 아스파라진(asparagine)으로의 변이, 232번째 아미노산인 시스테인(cysteine)의 세린(serine)으로의 변이, 357번째 아미노산인 프롤린(proline)의 아스파라진(asparagine)으로의 변이, 및 359번째 아미노산인 세린(serine)의 트레오닌(threonine)으로의 변이로 구성된 군에서 선택된 1종 이상의 변이를 포함하는 것인, 제조 방법.
(a) A biological fluid comprising a persistent human growth hormone NexP-hGH protein fused with alpha-1 antitrypsin mutant (NexP) and human growth hormone (hGH) is mixed with a resin with anti-alpha-1 antitrypsin antibody fragment Applying to affinity affinity chromatography; And
(b) isolating a sustained growth hormone NexP-hGH protein having an isoelectric point (pI) of 5.2 or less according to a difference in isoelectric point (pI), said method comprising the steps of: The alpha-1 antitrypsin mutant (NexP) is a mutation of the ninth glutamine to asparagine of the wild type alpha-1 antitrypsin protein, a mutation of the 232th amino acid cysteine to serine , A mutation of the 357th amino acid proline to asparagine, and a 359th amino acid serine to threonine. , Manufacturing method.
The method of claim 3 wherein the method of producing the step (b) includes the step of elution with a tris (Tris) buffer solution containing 0 to 1000mM MgCl 2.
The method of claim 4, wherein the Tris buffer is from 0 to 200mM MgCl 2 The method of manufacturing the tris buffer to the solution containing the.
4. The method according to claim 3, wherein the step (a) comprises applying a sample containing the persistent human growth hormone NexP-hGH protein to an anion exchange resin chromatography or hydrophobic resin chromatography, Is applied to an affinity chromatography packed with a resin with an antibody fragment attached thereto.
4. The method of claim 3, wherein step (a) comprises sequentially applying a sample containing the persistent human growth hormone NexP-hGH protein to anion exchange resin chromatography and hydrophobic resin chromatography, Lt; RTI ID = 0.0 > affinity < / RTI > chromatography packed with a resin with an anti-tryptic antibody fragment attached thereto.
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| JP2015515950A JP6231559B2 (en) | 2012-06-05 | 2013-06-05 | Method for producing and purifying highly glycosylated sustained human growth hormone protein |
| PCT/KR2013/004985 WO2013183948A1 (en) | 2012-06-05 | 2013-06-05 | Highly glycosylated long-acting human growth hormone protein and production method for same |
| CN201380029894.0A CN104379598B (en) | 2012-06-05 | 2013-06-05 | Long-acting human growth hormone's albumen of high glycosylation and its production method |
| US14/406,192 US9902753B2 (en) | 2012-06-05 | 2013-06-05 | Method of purifying a long-acting human growth hormone |
| EP13800310.8A EP2857417B1 (en) | 2012-06-05 | 2013-06-05 | Highly glycosylated long-acting human growth hormone protein and production method for same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0734393B1 (en) | 1993-12-13 | 1999-04-07 | Genentech, Inc. | Method for purifying polypeptides |
| KR20100116558A (en) * | 2009-04-22 | 2010-11-01 | (주)알테오젠 | Fusion protein or fusion peptide with increased half life by keeping long-acting activity, and method for increasing half life using the same |
| US20110072526A1 (en) | 2003-09-30 | 2011-03-24 | Sterrenbeld Biotechnologie North America, Inc. | Process for producing exogenous protein in the milk of transgenic mammals and a process for purifying proteins therefrom |
| US20110288005A1 (en) | 2010-04-02 | 2011-11-24 | Amunix Operating Inc. a Delware Corporation | Alpha 1-antitrypsin compositions and methods of making and using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0734393B1 (en) | 1993-12-13 | 1999-04-07 | Genentech, Inc. | Method for purifying polypeptides |
| US20110072526A1 (en) | 2003-09-30 | 2011-03-24 | Sterrenbeld Biotechnologie North America, Inc. | Process for producing exogenous protein in the milk of transgenic mammals and a process for purifying proteins therefrom |
| KR20100116558A (en) * | 2009-04-22 | 2010-11-01 | (주)알테오젠 | Fusion protein or fusion peptide with increased half life by keeping long-acting activity, and method for increasing half life using the same |
| US20110288005A1 (en) | 2010-04-02 | 2011-11-24 | Amunix Operating Inc. a Delware Corporation | Alpha 1-antitrypsin compositions and methods of making and using same |
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