KR101211971B1 - Method for comparing and evaluating of the structure of biosimilars using nuclear magnetic resonance spectrometer - Google Patents

Method for comparing and evaluating of the structure of biosimilars using nuclear magnetic resonance spectrometer Download PDF

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KR101211971B1
KR101211971B1 KR1020100045240A KR20100045240A KR101211971B1 KR 101211971 B1 KR101211971 B1 KR 101211971B1 KR 1020100045240 A KR1020100045240 A KR 1020100045240A KR 20100045240 A KR20100045240 A KR 20100045240A KR 101211971 B1 KR101211971 B1 KR 101211971B1
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박성혁
강선미
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Abstract

본 발명은 핵자기공명 분광기를 이용한 동등생물의약품 구조의 비교 평가 방법에 관한 것으로서, 보다 상세하게는 전처리과정을 거치지 않고 동등생물의약품에 대하여 1차원 1H-NMR 또는 2차원 1H-13C HSQC 스펙트럼을 얻어 수행하는 동등생물의약품 구조의 비교 평가 방법에 관한 것이다.
본 발명은 시판되는 약물을 동위원소 치환이나 복잡한 전처리 과정 없이 짧은 시간 안에 직접적으로 분석하여 동등생물의약품 간의 미세한 구조적 차이 및 동일성 비교 평가가 가능하도록 한다. The present invention relates to a method for comparative evaluation of the equivalent biological drug structure using nuclear magnetic resonance spectroscopy, more specifically, to obtain a one-dimensional 1H-NMR or two-dimensional 1H-13C HSQC spectrum for the equivalent biological drug without pretreatment It relates to a method for comparative evaluation of the structure of the equivalent biological drug product to be performed.
In the present invention, commercially available drugs can be directly analyzed in a short time without isotopic substitution or complex pretreatment, so that the microstructural differences and identity comparison between equivalent biological drugs can be evaluated.

Description

핵자기공명 분광기를 이용한 동등생물의약품 구조의 비교 평가 방법 {METHOD FOR COMPARING AND EVALUATING OF THE STRUCTURE OF BIOSIMILARS USING NUCLEAR MAGNETIC RESONANCE SPECTROMETER}Comparative evaluation method of equivalent biological drug structure using nuclear magnetic resonance spectroscopy {METHOD FOR COMPARING AND EVALUATING OF THE STRUCTURE OF BIOSIMILARS USING NUCLEAR MAGNETIC RESONANCE SPECTROMETER}

본 발명은 핵자기공명 분광기를 이용한 동등생물의약품 구조의 비교 평가 방법에 관한 것으로서, 보다 상세하게는 전처리과정을 거치지 않은 동등생물의약품 시료에 대하여 1차원 1H-NMR 또는 2차원 1H-13C HSQC 스펙트럼을 얻어 수행하는 동등생물의약품 구조의 비교 평가 방법에 관한 것이다.The present invention relates to a method for comparative evaluation of the structure of an equivalent biological drug using nuclear magnetic resonance spectroscopy, and more specifically, 1D 1H-NMR or 2D 1H-13C HSQC spectra for the equivalent biological drug sample that has not been pretreated. It relates to a method for comparative evaluation of the equivalent biological drug structure obtained and performed.

생물의약품이란 사람이나 다른 생물체에서 유래된 것을 원료 또는 재료로 하여 제조한 의약품으로 생물학적 제재, 유전자 재조합의약품, 세포배양의약품, 세포치료제, 유전자치료제 등을 포함한다. 생물의약품은 표적치료가 가능하며 치료효과가 높으나 비용이 많이 든다는 단점이 있다. 따라서 동등 생물의약품을 제조하여 오리지널 의약품보다 낮은 가격으로 동등한 수준의 제품을 공급함으로써 의료비 부담을 줄일 수 있다.Biopharmaceuticals are medicines manufactured from raw materials or materials derived from humans or other organisms, and include biological products, recombinant drugs, cell culture drugs, cell therapies, and gene therapies. Biopharmaceuticals have the disadvantage that they can be targeted and have high therapeutic effects but are expensive. Thus, the cost of medical expenses can be reduced by manufacturing equivalent biologics and supplying equivalent products at lower prices than the original.

다만, 생물의약품은 일반적으로 분자량이 크고 매우 복잡한 구조를 가진 단백질이므로 그 구조와 활성은 세포주의 종류와 제조 방법 변경에 매우 민감하며, 동일한 제조자가 동일한 제품을 제조할 때도 제조방법이 변경된다면 동일한 제품이 생산된다는 것을 보장할 수 없기 때문에 품질, 안전성, 유효성에 대한 동등성 평가가 이루어지고 있다.However, since biopharmaceuticals are generally proteins with high molecular weight and very complex structure, their structure and activity are very sensitive to changes in cell line type and manufacturing method. As it cannot be guaranteed that it is produced, equivalence assessments for quality, safety and effectiveness are being made.

동등생물의약품을 시판하기 위해서는 해당 단백질이 생체 내 단백질이나 이미 시판되고 있는 대조약과 구조적으로 유사하다는 것을 보여야 한다. 단백질의 구조는 궁극적으로 동등생물의약품의 질, 안정성 그리고 효능에 영향을 미친다. 따라서 최근 많은 생물의약품들이 연구되는 시점에 비슷한 효능을 가지는 것으로 추정되는 생물의약품들이 실제 구조적으로 얼마나 유사한지 판단할 수 있는 빠르고 정확한 시험법이 요구되고 있다.To market an equivalent biologic, the protein must be shown to be structurally similar to the protein in vivo or to a commercially available reference product. The structure of the protein ultimately affects the quality, stability and efficacy of the equivalent biologic. Therefore, there is a need for a fast and accurate test method that can determine how structurally similar biological drugs are expected to have similar efficacy at the time when many biological drugs are studied.

그러나 현재의 생물의약품의 단백질 구조 분석 및 비교는 생리활성 측정이나 HPLC (High Performance Liquid Chromatography)와 같은 간접적인 방법으로 이루어지고 있어, 돌연변이체와 같이 미세한 차이를 갖는 생물의약품의 분석에 적절하지 않다. However, current protein structure analysis and comparison of biopharmaceuticals is performed by indirect methods such as physiological activity measurement or HPLC (High Performance Liquid Chromatography), which is not suitable for analysis of biopharmaceuticals with small differences such as mutants.

최근 이러한 미세한 차이를 갖는 돌연변이체 동등생물의약품에 대한 구조적 성질을 평가하기 위하여 1H-15N HSQC (Heteronuclear single quantum coherence) 자기공명 분광법으로 단백질의 구조적 특성을 분석하는 방법이 발표되었다. Recently, in order to evaluate the structural properties of the mutant equivalent biopharmaceuticals having such minute differences, a method of analyzing the structural properties of proteins by 1H-15N heteronuclear single quantum coherence (HSQC) magnetic resonance spectroscopy has been published.

그러나 1H-15N HSQC의 방법은 15N 동위원소의 치환을 위한 시료의 전처리 과정이 필요하며 이러한 과정이 없이는 15N의 자연 존재비(natural abundance)가 낮아서 시판 중인 생물 의약품에 대한 적용은 현실적으로 불가능하다.However, the method of 1H-15N HSQC requires a pretreatment of a sample for the substitution of 15N isotopes, and without this process, the natural abundance of 15N is low, and thus it is practically impossible to apply to commercial biopharmaceuticals.

이에 본 발명은 상기의 문제점을 해결하고 상기의 필요성에 의하여 안출된 것으로서, 본 발명의 목적은 핵자기공명 분광기를 이용하여 단백질의 구조적 성질을 동위원소 치환 없이 직접적으로 분석함으로써 생물의약품 간의 미세한 구조적 차이 및 동일성을 비교 분석하는 방법을 제공함에 있다.The present invention has been made to solve the above problems and by the necessity of the above, an object of the present invention by using a nuclear magnetic resonance spectroscopy to directly analyze the structural properties of proteins without isotopic substitution microscopic structural differences between biologicals And a method for comparative analysis of identity.

상기의 목적을 달성하기 위하여, 본 발명은 핵자기공명 분광기를 이용한 동등생물의약품 구조의 비교 평가 방법을 제공한다.In order to achieve the above object, the present invention provides a comparative evaluation method of the equivalent biological drug structure using a nuclear magnetic resonance spectroscopy.

본 발명에서 동등생물의약품 구조의 비교 평가 방법은 생물의약품 시료를 그대로 준비하고, 상기 시료를 핵자기공명 분광기를 이용하여 분석하는 단계를 포함한다.In the present invention, the comparative evaluation method of the equivalent biological drug structure includes preparing a biopharmaceutical sample as it is, and analyzing the sample using a nuclear magnetic resonance spectrometer.

본 발명에 있어서, 상기 시료를 준비하는 단계는 희석이나 농축 과정을 생략하는 것을 특징으로 한다.In the present invention, the preparing of the sample is characterized in that the dilution or concentration process is omitted.

본 발명에 있어서, 상기 핵자기공명 분광기를 이용하여 분석하는 단계는 시료에 대하여 1차원 1H-NMR 스펙트럼 또는 2차원 1H-13C HSQC (Heteronuclear single quantum coherence) 스펙트럼을 얻어 수행하는 것을 특징으로 한다. 1차원 1H-NMR 스펙트럼을 얻고자 할 때는 30초 내지 3분 동안, 바람직하게는 1분 이내에 데이터를 얻고, 2차원 1H-13C HSQC 스펙트럼을 얻고자 할 때는 30분 내지 3시간 동안, 바람직하게는 2시간 이내에 데이터를 얻는 것을 특징으로 한다. In the present invention, the analyzing using the nuclear magnetic resonance spectrometer is characterized in that to perform a one-dimensional 1H-NMR spectrum or two-dimensional 1H-13C heteronuclear single quantum coherence (HSQC) spectrum for the sample. Data is obtained for 30 seconds to 3 minutes, preferably within 1 minute, for obtaining a one-dimensional 1H-NMR spectrum, and for 30 minutes to 3 hours, preferably for one second, for obtaining a two-dimensional 1H-13C HSQC spectrum. It is characterized by obtaining the data within two hours.

또한, 본 발명에 있어서 상기 동등생물의약품 구조의 비교 평가 방법은 인슐린 돌연변이체 개량생물의약품 구조의 비교 평가 방법인 것을 특징으로 하며, 상기 인슐린 돌연변이체 개량생물의약품은 글라진 또는 글루리신이다. In the present invention, the comparative evaluation method of the equivalent biologic drug structure is characterized in that the comparative evaluation method of the insulin mutant modified biologic drug structure, wherein the insulin mutant modified biologic drug is glargine or glulisine.

또한, 본 발명의 동등생물의약품 비교 평가 방법은 생물체에서 유래된 단백질 등을 원료 또는 재료로 하여 제조한 생물학적 제재, 유전자재조합 의약품, 세포배양 의약품, 세포 치료제 및 유전자 치료제 등에서 선택된 1종 이상의 단백질 생물의약품의 동등성을 비교 평가하는 데 유용하게 적용될 수 있으며, 이에 의해 제한되지 않는다.In addition, the comparative biopharmaceutical comparative evaluation method of the present invention is one or more protein biopharmaceuticals selected from biological products, genetically modified pharmaceuticals, cell culture pharmaceuticals, cell therapeutics and gene therapeutics prepared from proteins or the like derived from living organisms. Can be usefully applied to comparative evaluation of the equivalence of, without being limited thereto.

본 발명은 핵자기공명 분광법을 이용하여 단백질의 구조적 성질을 직접적으로 분석함으로써 동위원소 치환 없이 짧은 시간 안에 동등생물의약품 간의 미세한 구조적 차이 및 동일성 비교 평가가 가능하도록 한다. The present invention directly analyzes the structural properties of proteins using nuclear magnetic resonance spectroscopy, allowing for the comparison of minute structural differences and identity between equivalent biologics in a short time without isotopic substitution.

또한 본 발명은 시판되는 약물을 전처리가 없이 비파괴적으로 검사할 수 있으므로 농축 또는 희석 과정에서 생길 수 있는 단백질의 고차원적 구조의 변형을 막을 수 있을 뿐만 아니라 편리성과 유용성 측면에서도 많은 장점을 갖는다. In addition, the present invention can be tested non-destructively without the pre-treatment of the drug can not only prevent the modification of the high-dimensional structure of the protein that may occur during the concentration or dilution process, but also has many advantages in terms of convenience and usability.

도 1은 본 발명의 비교예에 따른 인슐린 글라진의 HPLC 크로마토그램을 나타낸 것이다.
도 2은 본 발명의 비교예에 따른 인슐린 글루리신의 HPLC 크로마토그램을 나타낸 것이다.
도 3은 본 발명의 일실시예에 따른 인슐린 글라진의 1차원 1H-NMR 스펙트럼을 나타낸 것이다.
도 4는 본 발명의 일실시예에 따른 인슐린 글루리신의 1차원 1H-NMR 스펙트럼을 나타낸 것이다.
도 5은 본 발명의 일실시예에 따른 인슐인 글라진의 2차원 1H-13C HSQC 스펙트럼을 나타낸 것이다.
도 6는 본 발명의 일실시예에 따른 인슐린 글루리신의 2차원 1H-13C HSQC 스펙트럼을 나타낸 것이다.1 shows an HPLC chromatogram of insulin glargine according to a comparative example of the present invention.
Figure 2 shows an HPLC chromatogram of insulin glulisine according to a comparative example of the present invention.
Figure 3 shows a one-dimensional 1H-NMR spectrum of insulin glargine according to an embodiment of the present invention.
Figure 4 shows a one-dimensional 1H-NMR spectrum of insulin glulisine according to an embodiment of the present invention.
Figure 5 shows a two-dimensional 1H-13C HSQC spectrum of the insulin glargine according to an embodiment of the present invention.
Figure 6 shows a two-dimensional 1H-13C HSQC spectrum of insulin glulisine according to an embodiment of the present invention.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 핵자기공명 분광기를 이용한 동등생물의약품 구조의 비교 평가 방법을 제공한다.The present invention provides a method for evaluating comparative biological drug structures using nuclear magnetic resonance spectroscopy.

구체적으로, 본 발명은 생물의약품 시료를 각각 준비하고, 상기 시료를 핵자기공명 분광기를 이용하여 분석하는 단계를 포함하는 것을 특징으로 한다.Specifically, the present invention is characterized in that it comprises the step of preparing a biopharmaceutical sample, respectively, and analyzing the sample using a nuclear magnetic resonance spectrometer.

본 발명의 상기 시료를 준비하는 단계는 생물의약품을 희석하거나 농축하는 전처리 과정 없이 시판되는 생물의약품을 그대로 NMR tube에 넣고 5vol%의 D2O를 첨가하여 측정하는 것을 특징으로 한다. Preparing the sample of the present invention is characterized in that by measuring the addition of 5vol% D2O into the NMR tube of the commercially available biopharmaceuticals without pretreatment process to dilute or concentrate the biopharmaceuticals.

본 발명에 있어서 상기 핵자기공명 분광기를 이용하여 분석하는 단계는 시료에 대하여 1차원 1H-NMR 스펙트럼 또는 2차원 1H-13C HSQC (Heteronuclear single quantum coherence) 스펙트럼을 얻어 수행하는 것을 특징으로 한다.
In the present invention, the analyzing using the nuclear magnetic resonance spectrometer is characterized in that the sample is obtained by performing a one-dimensional 1H-NMR spectrum or a two-dimensional 1H-13C heteronuclear single quantum coherence (HSQC) spectrum.

본 발명의 2차원 1H-13C HSQC 스펙트럼을 이용한 방법은 13C의 자연 존재비(natural abundance)를 이용한다. 기존에 단백질의 2차원 스펙트럼을 측정할 때 많이 사용하는 1H-15N HSQC는 15N 동위원소(isotope)의 치환(labeling)을 위하여 E.coli에서 재조합 단백질의 발현과정을 거쳐야 한다. 하지만 본 발명에서는 13C의 자연 존재비를 이용하므로 특별한 동위원소의 치환이 요구되지 않는다. 따라서 시판 중인 약품에 대한 1H-13C HSQC 스펙트럼을 직접적으로 분석할 수 있고, 이를 통하여 30분 내지 3시간 안에 동등생물의약품의 고차원 구조에 대한 정밀한 분석에 이용할 수 있다. 즉, 시판 중인 생물의약품에 대한 직접적인 적용이 가능하다. 그에 비하여 기존의 1H-15N HSQC는 13C 보다 자연 존재비가 낮은 15N을 사용하기 때문에 수십 시간 이상이 소모되므로 동위원소 치환이 되지 않은 시판 중인 상품에 대해 직접 적용하기는 현실적으로 불가능하다.
The method using the two-dimensional 1H-13C HSQC spectrum of the present invention utilizes a natural abundance of 13C. 1H-15N HSQC, which is widely used to measure two-dimensional spectra of proteins, has to undergo expression of recombinant proteins in E. coli for labeling of 15N isotopes. However, the present invention utilizes a natural abundance of 13 C, so no special isotope substitution is required. Therefore, the 1H-13C HSQC spectrum of the commercially available drug can be directly analyzed, which can be used for precise analysis of the high dimensional structure of the equivalent biological drug within 30 minutes to 3 hours. That is, direct application to commercial biologics is possible. In comparison, the conventional 1H-15N HSQC uses 15N, which has a lower natural abundance than 13C, which consumes more than a few tens of hours, making it practically impossible to apply directly to commercially available products that are not isotopically substituted.

이하, 본 발명의 구체적인 내용을 구체적인 비교예와 실험예를 들어 상세히 설명하고자 한다. 이들 실험예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 권리범위는 이들 실험예에만 한정되는 것은 아니다.
Hereinafter, specific contents of the present invention will be described in detail with reference to specific comparative examples and experimental examples. These experimental examples are only for illustrating the present invention, and the scope of the present invention is not limited only to these experimental examples.

비교예 1. 인슐린 글라진과 인슐린 글루리신에 대한 HPLC 실시Comparative Example 1. HPLC of Insulin Glargine and Insulin Glycine

본 발명의 일실시예와 비교분석을 위해 인슐린 돌연변이체 개량생물의약품인 인슐린 글라진과 인슐린 글루리신에 대한 HPLC를 시행하였다. 시료는 시판 중인 인슐린 글루리신과 인슐린 글라진을 각각 20배로 희석시켜서 준비하여 사용하였다. A 용액은 0.1vol%의 포름산(formic acid)을 포함하는 증류수를 사용하고 B 용액은 0.1vol%의 포름산(formic acid)을 포함하는 아세토니트릴(CH3CN)을 사용하였다. 두 용액의 농도 구배는 B용액의 농도를 40%에서 80%까지 (이때 A용액의 농도는 60%에서 20%이다) 20분간 선형으로 (linear) 올려주고, B 용액 100%에서 5분간, A 용액 100%에서 5분간, 총 30분간 0.2 ㎖/min의 유속으로 진행하였다. 그리고 신호는 UV 210 ㎚로 검출 되었다. 이때 사용된 기기는 Gilson사의 802C 모델이며, 사용된 컬럼은 Gemini-NX3u C18이다.For comparative analysis with one embodiment of the present invention, HPLC was performed for insulin glargine and insulin glycerin, which are insulin mutant-modified biologics. Samples were prepared by diluting each of commercially available insulin glycine and insulin glargine by 20-fold. The solution A used distilled water containing 0.1 vol% formic acid and the solution B used acetonitrile (CH 3 CN) containing 0.1 vol% formic acid. The concentration gradient of the two solutions raises the concentration of solution B from 40% to 80% (the concentration of solution A is from 60% to 20%) for 20 minutes, and for 5 minutes at B solution 100% The solution was run at 100% solution for 5 minutes at a flow rate of 0.2 ml / min for 30 minutes. And the signal was detected with UV 210 nm. The instrument used was a Gilson 802C model and the column used was a Gemini-NX3u C18.

도 1과 도 2는 시판되고 있는 인슐린 글라진과 인슐린 글루리신의 HPLC 결과이다. 두 인슐린이 서로 비슷한 시간대에 용출 (elution) 되어 기존의 HPLC 방법으로는 구분이 어려움을 알 수 있다.
1 and 2 are HPLC results of the commercially available insulin glargine and insulin globulin. The two insulins are eluted at similar times, which makes it difficult to distinguish between conventional HPLC methods.

실시예 1. 인슐린 글라진과 인슐린 글루리신에 대한 1차원 1H-NMR 실시Example 1 One-Dimensional 1H-NMR for Insulin Glargine and Insulin Glycine

본 발명의 인슐린 글라진과 인슐린 글루리신의 1차원 1H-NMR 스펙트럼을 얻기 위해 각각의 인슐린 샘플을 희석하거나 농축하는 전처리 과정 없이 그대로 NMR 튜브에 넣고 5vol% 의 D2O를 첨가한 후 Bruker 900 MHz에서 Cryoprobe를 사용하여 1분 여 동안 1차원 1H-NMR 데이터를 얻었다. 스펙트럼의 넓이는 1H 축에 대하여 12626.263 Hz로 측정하였다. In order to obtain the 1-dimensional 1H-NMR spectra of the insulin glargine and insulin glulisine of the present invention, each insulin sample was added to the NMR tube without addition or diluting, and 5 vol% of D2O was added to the Cryoprobe at Bruker 900 MHz. One-dimensional 1H-NMR data was obtained for about one minute using. The width of the spectrum was measured at 12626.263 Hz about the 1H axis.

도 3과 도 4는 시판되고 있는 인슐린 글라진과 인슐린 글루리신으로부터 1차원 1H-NMR 스펙트럼을 얻은 결과이다. 두 제품은 인슐린과 단지 2개의 아미노산이 다른 돌연변이체임에도 불구하고 1차원 스펙트럼 양상이 확연히 다름을 확인할 수 있다. FIG. 3 and FIG. 4 show the results of one-dimensional 1H-NMR spectra from commercially available insulin glargine and insulin glycerin. Although the two products are different mutants from insulin and only two amino acids, one-dimensional spectral patterns are clearly different.

인슐린 글라진의 경우 폭이 좁은 신호가 매우 많이 나오는 것을 알 수 있고, 인슐린 글루리신의 경우 신호의 폭이 매우 넓어 신호 간의 중첩이 심하며 신호의 개수가 현저히 적다.In the case of insulin glargine, it can be seen that a very narrow signal comes out. In the case of insulin glucinine, the signal is very wide, so there is a great overlap between signals and the number of signals is very small.

일반적으로 사용되는 HPLC 방법으로 인슐린 글라진과 글루리신을 분석한 도 1과 도 2에 거의 차이가 없다는 것을 근거로, 30분 이상 소요되는 HPLC 방법에 비하여 본 발명에서의 방법이 매우 빠른 시간에 복잡한 시료의 전처리 과정 없이 간단하고 확실하게 두 생물의약품의 차이를 구분할 수 있는 데이터를 제공함을 알 수 있다. 뿐만 아니라 1분여의 짧은 시간이 소요되므로 오랜 측정 시간에 따른 단백질의 변성이나 HPLC에 많이 사용되는 유기용매에 따른 인위결과(artifact)에 관한 문제의 가능성을 배제시킬 수 있다.
The method of the present invention is complicated in a very fast time compared to the HPLC method which takes 30 minutes or more, based on the fact that there is almost no difference between FIGS. 1 and 2 for analyzing insulin glargine and glulisine by the commonly used HPLC method. It can be seen that it provides data to distinguish between the two biologicals simply and reliably without sample pretreatment. In addition, the short time of about 1 minute can eliminate the possibility of problems related to the degeneration of the protein according to a long measurement time or the artifacts due to the organic solvents used in HPLC.

실시예 2. 인슐린 글라진과 글루리신에 대한 2차원 1H-13C HSQC 실시Example 2 Two-Dimensional 1H-13C HSQC Implementation of Insulin Glargine and Glycine

구조의 완결성에 대한 더 정밀한 분석은 2차원 1H-13C HSQC (hetero nuclear single quantum correlation) 방법을 사용할 수 있다. 상기 실시예 1에서와 같이 준비한 시료를 가지고 2차원 1H-13C HSQC NMR 실험을 수행하였다. 이때, Bruker 900 MHz에서 Cryoprobe를 이용하였다. 스펙트럼 넓이는 1H, 13C 축에 대하여 각각 14423.077 Hz와 13586.957 Hz로 측정하였다. 90˚ 펄스의 길이는 1H, 13C에 대하여 각각 17.4 μsec, 11.8 μsec였다. 13C 디커플링은 acquisition동안에 GARP를 사용하였고, t1 측정의 프로세싱은 Echo/Antiecho-TPPI gradient selection 방법으로 흡수모드의 스펙트럼을 얻었다. 콤플렉스 데이터 매트릭스는 128(t1) * 1024(t2) 포인트를 측정하였다. A more precise analysis of the structural integrity can be achieved using the two-dimensional 1H-13C hetero nuclear single quantum correlation (HSQC) method. Two-dimensional 1H-13C HSQC NMR experiments were performed with the samples prepared as in Example 1. At this time, Cryoprobe was used at Bruker 900 MHz. The spectral width was measured at 14423.077 Hz and 13586.957 Hz for the 1H and 13C axes, respectively. The lengths of the 90 ° pulses were 17.4 μsec and 11.8 μsec for 1H and 13C, respectively. 13C decoupling used GARP during acquisition, and the processing of t1 measurements obtained the spectrum of absorption mode by Echo / Antiecho-TPPI gradient selection method. The complex data matrix measured 128 (t1) * 1024 (t2) points.

2차원 1H-13C HSQC 스펙트럼을 얻기 위해 1 시간 동안 실험하여 얻은 시간 영역의 데이터를 푸리에 변환(Fourier Transform)하여 주파수 영역으로 데이터를 변환하였다.In order to obtain a two-dimensional 1H-13C HSQC spectrum, Fourier transform of the data in the time domain obtained by experimenting for one hour was performed to transform the data into the frequency domain.

도 5와 도 6에서 인슐린 글라진과 인슐린 글루리신에 대한 2차원 1H-13C HSQC NMR의 비교분석 데이터를 나타내었다. 5 and 6 show comparative analysis data of two-dimensional 1H-13C HSQC NMR for insulin glargine and insulin glycerin.

본 발명의 일실시예에서 인슐린 글라진과 인슐린 글루리신의 농도는 같음에도 불구하고 도 5의 2차원 1H-13C HSQC 스펙트럼 신호는 도 6의 그것보다 훨씬 많이 대각선 방향으로 고루 퍼져 있는 것을 볼 수 있다. 2차원 핵자기공명 스펙트럼 피크의 이러한 양상은 두 생물의약품의 고차원 구조가 다르다는 것을 확실히 밝혀 준다. In one embodiment of the present invention, the two-dimensional 1H-13C HSQC spectral signal of FIG. 5 is spread evenly in a diagonal direction even more than that of FIG. . This aspect of the two-dimensional nuclear magnetic resonance spectral peak clearly shows that the high-dimensional structures of the two biologicals are different.

Claims (7)

삭제delete 삭제delete 삭제delete 생물의약품 시료를 동위원소 표지 없는 그대로 핵자기공명 분광기를 이용하여 상기 시료에 대해 30분 내지 3시간 동안 2차원 1H-13C HSQC (Heteronuclear single quantum coherence) 스펙트럼을 얻어 분석하는 단계를 포함하는 인슐린 돌연변이체 개량생물의약품 구조의 비교 평가 방법.
Insulin mutant comprising the step of obtaining a two-dimensional 1H-13C heteronuclear single quantum coherence (HSQC) spectrum for 30 minutes to 3 hours for the sample using a nuclear magnetic resonance spectroscopy without an isotopic label Comparative assessment of improved biologic structure.
삭제delete 삭제delete 제 4항에 있어서,
상기 인슐린 돌연변이체 개량생물의약품은 인슐린 글라진 또는 인슐린 글루리신인 것을 특징으로 하는 동등생물의약품 구조의 비교 평가 방법.5. The method of claim 4,
The insulin mutant ameliorating biological drug is a comparative evaluation method of the equivalent biopharmaceutical structure, characterized in that insulin glargine or insulin glulisine.
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