KR102204927B1 - Method for quality evaluation of capsular polysacchride-protein conjugate vaccine - Google Patents
Method for quality evaluation of capsular polysacchride-protein conjugate vaccine Download PDFInfo
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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Abstract
본 발명은 피막 다당류-단백질 접합 백신의 품질 평가 방법에 관한 것으로, 피막 다당류-접합 단백질 접합체에 포함된 유리당류의 함량을 측정함으로써, 제조된 접합 백신의 품질을 평가하는데 유용하게 사용할 수 있다.The present invention relates to a method for evaluating the quality of a capsular polysaccharide-protein conjugated vaccine, and can be usefully used to evaluate the quality of a prepared conjugated vaccine by measuring the content of free saccharides contained in the capsular polysaccharide-conjugated protein conjugate.
Description
본 발명은 피막 다당류-단백질 접합 백신의 품질 평가 방법에 관한 것이다.
The present invention relates to a method for evaluating the quality of a capsular polysaccharide-protein conjugated vaccine.
인체에서 병원성을 나타내는 주요 항원 물질인 세균의 피막 다당류(capsular polysaccharide, CPS)는, 세균의 감염을 예방하기 위한 백신 항원으로 사용된다. 그러나, 상기 CPS를 이용하여 제조된 백신을 단독으로 투여하는 경우 3년 이하의 짧은 면역기간을 보이며, 특히 2세 이하의 유아에서 낮은 면역원성을 나타낸다. 따라서, 이러한 한계를 극복하기 위하여 CPS에 단백질을 접합한 다당류-단백질 접합 백신이 개발되었다.Bacterial capsular polysaccharide (CPS), which is a major antigenic substance showing pathogenicity in the human body, is used as a vaccine antigen to prevent bacterial infection. However, when the vaccine prepared using the CPS is administered alone, a short immunity period of 3 years or less is shown, and particularly, low immunogenicity is shown in infants 2 years or younger. Therefore, in order to overcome this limitation, a polysaccharide-protein conjugation vaccine was developed in which a protein was conjugated to CPS.
다당류-단백질 접합 백신은, 제조 과정에서 생성된 다당류 및 단백질 접합체의 접합율, 결합되지 않은 유리당류의 비율, 및 형성된 접합체의 크기 등에 의해 그 품질을 평가할 수 있다.The quality of the polysaccharide-protein conjugated vaccine can be evaluated by the conjugation rate of the polysaccharide and protein conjugate produced in the manufacturing process, the ratio of unbound free saccharide, and the size of the formed conjugate.
이와 관련하여, 대한민국 등록특허 제10-0947757호는 다가 수막구균 다당류-단백질 접합체 백신의 제조방법을 개시하고 있으나, 상기 제조된 접합체 백신의 품질을 평가하는 방법은 언급하고 있지 않다.
In this regard, Korean Patent Registration No. 10-0947757 discloses a method for preparing a multivalent meningococcal polysaccharide-protein conjugate vaccine, but does not mention a method for evaluating the quality of the prepared conjugate vaccine.
한편, 유리당류는 다당류-단백질 접합 백신의 제조 과정에서 접합 단백질과 접합체를 형성하지 않은 다당류를 의미한다. 일반적으로, 접합 백신에 포함된 유리당류의 함량이 낮을수록 상기 백신의 면역원성이 좋은 것으로 알려져 있다.Meanwhile, free saccharide refers to a polysaccharide that does not form a conjugate with a conjugated protein in the manufacturing process of a polysaccharide-protein conjugated vaccine. In general, it is known that the lower the content of free saccharides contained in the conjugated vaccine, the better the immunogenicity of the vaccine.
이와 같은 유리당류의 함량은, 접합 백신에 포함된 유리당류를 정제 및 정량하고, 정량한 유리당류의 함량을 반응에 사용된 총 당류의 함량과 비교하여 결정된다. 접합 백신에서 유리당류의 함량을 측정하기 위한 종래의 방법으로는 수산화알루미늄 젤을 이용한 단백질 흡착법, 디옥시콜레이트(deoxycholate)를 이용한 단백질 침전법 등이 알려져 있다.
The content of such free saccharides is determined by purifying and quantifying the free saccharides contained in the conjugated vaccine, and comparing the quantified amount of free saccharides with the content of total saccharides used in the reaction. Conventional methods for measuring the content of free saccharides in conjugated vaccines include a protein adsorption method using aluminum hydroxide gel and a protein precipitation method using deoxycholate.
본 발명의 목적은 피막 다당류-단백질 접합 백신의 품질 평가 방법을 제공하는 것이다.
An object of the present invention is to provide a method for evaluating the quality of a capsular polysaccharide-protein conjugated vaccine.
상기 목적을 달성하기 위하여, 본 발명은 피막 다당류-접합 단백질의 접합체에 소수성을 부여하는 단계; 소수성이 부여된 접합체로부터 유리당류를 분리하는 단계; 및 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법을 제공한다.In order to achieve the above object, the present invention comprises the steps of imparting hydrophobicity to the conjugate of the capsular polysaccharide-conjugated protein; Separating the free saccharide from the conjugate to which hydrophobicity is imparted; And it provides a method for evaluating the quality of the capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide.
또한, 본 발명은 피막 다당류-접합 단백질의 접합 백신에 소수성을 부여하는 단계; 소수성이 부여된 접합 백신으로부터 유리당류를 분리하는 단계; 및 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법을 제공한다.
In addition, the present invention comprises the steps of imparting hydrophobicity to a conjugated vaccine of a capsular polysaccharide-conjugated protein; Separating the free saccharide from the conjugated vaccine imparted with hydrophobicity; And it provides a method for evaluating the quality of the capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide.
본 발명은 피막 다당류-접합 단백질 접합체에 포함된 유리당류의 함량을 측정함으로써 제조된 접합 백신의 품질을 평가하는데 유용하게 사용할 수 있다.
The present invention can be usefully used to evaluate the quality of the prepared conjugated vaccine by measuring the content of free saccharides contained in the capsular polysaccharide-conjugated protein conjugate.
도 1은 장티푸스균의 피막 다당류를 사용하여 HPAEC-PAD를 수행한 결과, 용출 완충액의 농도구배에 따라 용출되는 다당류의 피크(peak)가 분리되는 것을 보여주는 그래프이다.
도 2는 유리당류 분획(A), 또는 장티푸스균의 피막 다당류 및 디프테리아 톡소이드 접합 단백질의 접합체 분획(B)에 포함되는 유리당류의 함량을 측정하기 위해 HPAEC-PAD를 수행한 결과를 나타내는 그래프이다.
도 3은 장티푸스균의 피막 다당류-단백질 접합체를 사용하여 소수성 상호작용 크로마토그래피를 하였을 때, 접합 단백질(DT 단독), 피막 다당류(Vi 단독), 이의 혼합물(DT+Vi) 또는 피막 다당류-단백질 접합체의 피크를 나타낸 결과 그래프이다.1 is a graph showing that the peak of the eluted polysaccharide is separated according to the concentration gradient of the elution buffer as a result of performing HPAEC-PAD using the capsular polysaccharide of typhoid bacteria.
Figure 2 is a graph showing the results of performing HPAEC-PAD to measure the content of free saccharides contained in the free saccharide fraction (A) or the conjugate fraction (B) of the capsular polysaccharide of typhoid bacteria and diphtheria toxoid conjugated protein.
3 is a conjugated protein (DT only), a capsular polysaccharide (Vi only), a mixture thereof (DT+Vi) or a capsular polysaccharide-protein conjugate when hydrophobic interaction chromatography was performed using the capsular polysaccharide-protein conjugate of Typhoid bacteria. It is a result graph showing the peak of.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 피막 다당류-접합 단백질의 접합체에 소수성을 부여하는 단계; 소수성이 부여된 접합체로부터 유리당류를 분리하는 단계; 및 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법을 제공한다.The present invention comprises the steps of imparting hydrophobicity to the conjugate of the capsular polysaccharide-conjugated protein; Separating the free saccharide from the conjugate to which hydrophobicity is imparted; And it provides a method for evaluating the quality of the capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide.
본 명세서에서 사용된 용어 "피막 다당류-접합 단백질의 접합체"는 접합 백신을 제조하기 위해 피막 다당류 및 접합 단백질을 접합시킨 접합체를 의미한다. 상기 접합체를 통상적인 방법으로 제형화하여 피막 다당류-단백질 접합 백신을 제조할 수 있다. 따라서, 본 발명의 품질 평가 방법은 피막 다당류 및 접합 단백질의 접합체가 포함된 접합 백신의 품질 평가에 사용할 수 있다.The term "conjugate of a capsular polysaccharide-conjugated protein" as used herein refers to a conjugate obtained by conjugating a capsular polysaccharide and a conjugated protein to prepare a conjugated vaccine. The conjugate can be formulated in a conventional manner to prepare a capsular polysaccharide-protein conjugated vaccine. Therefore, the quality evaluation method of the present invention can be used to evaluate the quality of a conjugated vaccine containing a conjugate of a capsular polysaccharide and a conjugated protein.
본 발명의 피막 다당류는 세균성 피막 다당류일 수 있고, 상기 세균성 피막 다당류는 폐렴구균(Streptococcus pneumoniae) 피막 다당류, 수막구균(Neisseria meningitidis) 피막 다당류, 장티푸스균(Salmonella typhi) 피막 다당류, 해모필루스 인플루엔자 B형(Haemophilus influenza B) 피막 다당류 및 이의 조합으로 구성된 군으로부터 선택될 수 있다. 본 발명의 일 실시예에서, 상기 세균성 피막 다당류는 장티푸스균 피막 다당류 또는 수막구균 피막 다당류일 수 있다.The capsular polysaccharide of the present invention may be a bacterial capsular polysaccharide, and the bacterial capsular polysaccharide is Streptococcus pneumoniae capsular polysaccharide, Neisseria meningitidis capsular polysaccharide, Salmonella typhi capsular polysaccharide, Haemophilus influenza B Type ( Haemophilus influenza B) capsular polysaccharides and combinations thereof. In one embodiment of the present invention, the bacterial capsular polysaccharide may be a typhoid capsular polysaccharide or a meningococcal capsular polysaccharide.
상기 수막구균 피막 다당류는 혈청 그룹 A, C, W135 또는 Y일 수 있다.The meningococcal capsular polysaccharide may be serogroup A, C, W135 or Y.
본 발명의 접합 단백질은 디프테리아 톡소이드, 디프테리아 톡소이드 무독화 변이체 CRM197, 테타누스 톡소이드 및 이의 조합으로 구성된 군으로부터 선택될 수 있다. 본 발명의 일 실시예에서, 상기 접합 단백질은 디프테리아 톡소이드 또는 디프테리아 톡소이드 무독화 변이체 CRM197일 수 있다. 상기 접합 단백질의 폴리뉴클레오티드 서열은 통상의 기술분야에 알려진 것이면 모두 사용할 수 있다.The conjugated protein of the present invention may be selected from the group consisting of diphtheria toxoid, diphtheria toxoid detoxifying variant CRM197, tetanus toxoid, and combinations thereof. In one embodiment of the present invention, the conjugated protein may be diphtheria toxoid or diphtheria toxoid detoxifying variant CRM197. Any polynucleotide sequence of the conjugated protein can be used as long as it is known in the art.
본 발명의 품질 평가 방법은 유리당류 및 피막 다당류-접합 단백질 접합체가 염의 첨가에 의한 소수성 차이에 의해 분리되는 것을 이용한다. 이때, 상기 유리당류는 낮은 소수성을 나타내고, 접합 단백질은 높은 소수성을 나타낸다.In the quality evaluation method of the present invention, the free saccharide and the capsular polysaccharide-conjugated protein conjugate are separated by a difference in hydrophobicity due to the addition of a salt. At this time, the free saccharide exhibits low hydrophobicity, and the conjugated protein exhibits high hydrophobicity.
본 발명의 방법에서 상기 염은 황산 암모늄, 황산칼륨, 황산나트륨, 염화나트륨 및 이의 조합으로 구성된 군으로부터 선택될 수 있다. 본 발명의 일 실시예에서, 상기 염은 황산 암모늄일 수 있다. 상기 염의 농도는 0.5 내지 2 M, 0.7 내지 1.8 M, 1.0 내지 1.7 M일 수 있다. 본 발명의 일 실시예에서, 상기 염의 농도는 1.0 또는 1.7 M일 수 있다.In the method of the present invention, the salt may be selected from the group consisting of ammonium sulfate, potassium sulfate, sodium sulfate, sodium chloride, and combinations thereof. In one embodiment of the present invention, the salt may be ammonium sulfate. The concentration of the salt may be 0.5 to 2 M, 0.7 to 1.8 M, 1.0 to 1.7 M. In one embodiment of the present invention, the concentration of the salt may be 1.0 or 1.7 M.
본 발명의 방법에서, 상기 분리는 소수성 상호작용 크로마토그래피(hydrophobic interaction chromatography) 또는 수산화알루미늄 흡착법을 이용하여 수행될 수 있다. 상기 분리는 접합 백신의 제조에 사용된 다당류와 접합 단백질의 종류에 따라 적절한 방법으로 수행될 수 있다.In the method of the present invention, the separation may be performed using hydrophobic interaction chromatography or an aluminum hydroxide adsorption method. The separation may be performed by an appropriate method depending on the type of polysaccharide and conjugated protein used in the preparation of the conjugated vaccine.
상기 소수성 상호작용 크로마토그래피는, 페닐, 옥틸, 부틸 및 이의 조합으로 구성된 군으로부터 선택되는 리간드가 결합된 수지가 충전된 컬럼을 이용하여 수행될 수 있다. 본 발명의 일 실시예에서, 상기 리간드는 페닐일 수 있다. 또한, 상기 수지는 아가로즈, 폴리스티렌, 다이비닐, 벤젠 및 이의 조합으로 구성된 군으로부터 선택되는 어느 하나일 수 있다. 본 발명의 일 실시예에서, 상기 수지는 아가로즈일 수 있다.The hydrophobic interaction chromatography may be performed using a column filled with a resin to which a ligand selected from the group consisting of phenyl, octyl, butyl, and combinations thereof is bound. In one embodiment of the present invention, the ligand may be phenyl. In addition, the resin may be any one selected from the group consisting of agarose, polystyrene, divinyl, benzene, and combinations thereof. In one embodiment of the present invention, the resin may be agarose.
본 발명의 리간드는 상기 피막 다당류-접합 단백질의 접합체에 소수성을 부여하기 위해 첨가하는 염을 사용하여 평형화될 수 있다. 이때, 소수성 상호작용 크로마토그래피에서 수지에 적용되는 접합체의 농도는 0.2 내지 2.0 ㎎/㎖일 수 있다.The ligand of the present invention can be equilibrated using a salt added to impart hydrophobicity to the conjugate of the capsular polysaccharide-conjugated protein. In this case, the concentration of the conjugate applied to the resin in hydrophobic interaction chromatography may be 0.2 to 2.0 mg/ml.
상기 유리당류의 함량 측정은 HPAEC-PAD(high performance anion exchange chromatography pulsed with amperometric detection)를 사용하여 수행될 수 있다.
The measurement of the free sugar content may be performed using high performance anion exchange chromatography pulsed with amperometric detection (HPAEC-PAD).
또한, 본 발명은 피막 다당류-접합 단백질의 접합 백신에 소수성을 부여하는 단계; 소수성이 부여된 접합 백신으로부터 유리당류를 분리하는 단계; 및 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법을 제공한다.In addition, the present invention comprises the steps of imparting hydrophobicity to a conjugated vaccine of a capsular polysaccharide-conjugated protein; Separating the free saccharide from the conjugated vaccine imparted with hydrophobicity; And it provides a method for evaluating the quality of the capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide.
상기 접합 백신의 품질 평가 방법은 상술한 특징을 가질 수 있다.
The method for evaluating the quality of the conjugated vaccine may have the above-described characteristics.
이하, 본 발명을 하기 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명이 이들에 의해 제한되는 것은 아니다.
Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are for illustrative purposes only, and the present invention is not limited thereto.
실시예Example 1. 장티푸스균의 피막 다당류-디프테리아 1. The capsular polysaccharide of typhoid bacteria-diphtheria 톡소이드Toxoid 접합체 및 백신의 제조 Preparation of conjugates and vaccines
문헌 [Kothari S. et al., Vaccine, 31(42):4714-4719(2013)]을 참조하여 장티푸스균의 피막 다당류를 정제하였다. 정제된 피막 다당류와 디프테리아 톡소이드 접합 단백질을 사용하여 공지된 방법으로 장티푸스균의 피막 다당류-디프테리아 톡소이드 접합체 및 상기 접합체를 포함하는 백신을 제조하였다.
The capsular polysaccharide of typhoid bacteria was purified with reference to the literature [Kothari S. et al., Vaccine, 31(42):4714-4719(2013)]. Using the purified capsular polysaccharide and diphtheria toxoid conjugation protein, a capsular polysaccharide-diphtheria toxoid conjugate of typhoid bacteria and a vaccine containing the conjugate were prepared by a known method.
실시예Example 2. 2. 수막구균의Meningococcal 활성 다당류-디프테리아 Active polysaccharide-diphtheria 톡소이드Toxoid 무독화 Detoxification 변이체Variant CRM197 접합체의 제조 Preparation of CRM197 conjugate
대한민국 등록특허 제10-0947757호를 참조하여 수막구균의 활성 다당류-디프테리아 톡소이드 무독화 변이체 CRM197 접합체 및 상기 접합체를 포함하는 백신을 제조하였다.With reference to Korean Patent Registration No. 10-0947757, an active polysaccharide of meningococcal-diphtheria toxoid detoxifying variant CRM197 conjugate and a vaccine containing the conjugate were prepared.
그 결과, 혈청그룹 A, C, W135 또는 Y의 활성 다당류와 디프테리아 톡소이드 CRM197의 접합체 및 상기 접합체가 포함된 백신을 수득하였다.
As a result, a conjugate of active polysaccharide of serogroup A, C, W135 or Y and diphtheria toxoid CRM197 and a vaccine containing the conjugate were obtained.
실험예Experimental example
1. 장티푸스균의 피막 다당류-디프테리아 톡소이드 접합체의 유리당류 함량 측정 1. Measurement of free saccharide content of polysaccharide-diphtheria toxoid conjugate of typhoid bacteria
1-1. 접합체에 포함된 유리당류의 분리1-1. Separation of free sugars contained in conjugates
상기 실시예 1에서 제조한 장티푸스균의 피막 다당류-디프테리아 톡소이드 접합체에 포함된 유리당류 함량을 측정하기 위하여, 소수성 상호작용 크로마토그래피(hydrophobic interaction chromatography, HIC)를 사용하여 상기 접합체에서 유리당류를 분리하였다.In order to measure the content of free saccharides contained in the film polysaccharide-diphtheria toxoid conjugate of typhoid bacteria prepared in Example 1, free saccharides were separated from the conjugate using hydrophobic interaction chromatography (HIC). .
구체적으로, 소수성 수지로서 페닐 리간드를 가진 6% 가교 아가로즈 수지(페닐 HP 수지)(GE Healthcare, 미국)가 충전된 1.6x10 ㎝의 폴리프로필렌 컬럼을 준비하였다. 여기에 평형화 완충액(1.7 M의 황산암모늄을 포함하는 50 mM 트리스, pH 7.5)을 1 내지 2 ㎖/분의 속도로 흘려보내 컬럼을 평형화시켰다.Specifically, a 1.6x10 cm polypropylene column filled with a 6% crosslinked agarose resin (phenyl HP resin) (GE Healthcare, USA) having a phenyl ligand as a hydrophobic resin was prepared. The column was equilibrated by flowing an equilibration buffer (50 mM Tris containing 1.7 M ammonium sulfate, pH 7.5) at a rate of 1 to 2 ml/min.
한편, 장티푸스균 피막 다당류 접합체를 동량의 평형화 완충액과 혼합하여 시료를 준비하였다. 백신도 상기와 동일하게 시료를 준비하였다. 준비된 시료들을 상기 평형화된 컬럼에 각각 로딩한 후, 소수성 수지에 결합되지 않고 용출되는 유리당류 분획을 수득하였다. 또한, 로딩 후, 용출 완충액(0.9% 생리식염수)을 1 내지 2 ㎖/분의 유속으로 가하여 소수성 수지에 접합된 접합체를 용출시켜 접합체 분획을 수득하였다.
On the other hand, a sample was prepared by mixing the typhoid bacteria capsular polysaccharide conjugate with an equal amount of equilibration buffer. Samples were prepared in the same manner as described above. After the prepared samples were each loaded onto the equilibrated column, a free saccharide fraction eluted without being bound to a hydrophobic resin was obtained. Further, after loading, an elution buffer (0.9% physiological saline) was added at a flow rate of 1 to 2 ml/min to elute the conjugate conjugated to the hydrophobic resin to obtain a conjugate fraction.
1-2. 분리된 유리당류의 함량 측정1-2. Measurement of the content of separated free sugars
실험예 1-1에서 수득한 각 분획에 포함되는 유리당류의 함량을 측정하기 위해 페닐 리간드를 갖는 PA1 컬럼(Dionex 사, 미국)을 사용하여 HPAEC-PAD(high performance anion exchange chromatography pulsed with amperometric detection)를 수행하였다.HPAEC-PAD (high performance anion exchange chromatography pulsed with amperometric detection) using a PA1 column having a phenyl ligand (Dionex, USA) to measure the content of free saccharides contained in each fraction obtained in Experimental Example 1-1 Was performed.
먼저, 용출 완충액의 농도구배에 따른 다당류 피크의 분리를 통해 HPAEC-PAD 방법이 유리당류의 정량에 적합한지 여부를 확인하였다. 구체적으로, 상기 접합체 분획에 2 M의 농도가 되도록 수산화나트륨을 첨가하고 110℃에서 4시간 동안 가열하여 상기 분획에 포함된 다당류를 가수분해시켰다. 가수분해된 시료는 0.2 ㎛의 필터로 여과하여 준비하였다. 한편, 상기 컬럼은 1 내지 2 ㎖/분의 유속으로 평형화 완충액(0.01 내지 0.2 M 수산화나트륨)을 가하여 평형화시켰다. 상기 준비된 시료를 평형화된 컬럼에 로딩한 뒤, 용출 완충액으로서 100 내지 200 mM의 질산나트륨을 포함하는 수산화나트륨 용액을 이용하여 용출 분획을 수득한 결과를 도 1에 나타내었다. 이때, 상기 용출 완충액은 하기 표 1에 기재된 바와 같은 농도구배 조건으로 1 ㎖/분의 유속으로 가하였다.First, it was confirmed whether the HPAEC-PAD method was suitable for quantification of free saccharides by separating the polysaccharide peaks according to the concentration gradient of the elution buffer. Specifically, sodium hydroxide was added to the conjugate fraction to a concentration of 2 M, and heated at 110° C. for 4 hours to hydrolyze the polysaccharide contained in the fraction. The hydrolyzed sample was prepared by filtering through a 0.2 μm filter. Meanwhile, the column was equilibrated by adding an equilibration buffer (0.01 to 0.2 M sodium hydroxide) at a flow rate of 1 to 2 ml/min. After loading the prepared sample onto the equilibrated column, the result of obtaining an eluted fraction using a sodium hydroxide solution containing 100 to 200 mM sodium nitrate as an elution buffer is shown in FIG. 1. At this time, the elution buffer was added at a flow rate of 1 ml/min under the conditions of the concentration gradient as shown in Table 1 below.
도 1에 나타난 바와 같이, 용출액의 농도구배에 따라 다당류의 피크가 잘 분리되는 것을 확인하였다.As shown in Figure 1, it was confirmed that the peak of the polysaccharide was well separated according to the concentration gradient of the eluate.
상기와 동일한 방법을 사용하여 실험예 1-1에서 수득한 각 분획을 HPAEC-PAD로 분석하고 유리당류의 함량을 측정하였다.Using the same method as described above, each fraction obtained in Experimental Example 1-1 was analyzed by HPAEC-PAD, and the content of free saccharides was measured.
그 결과, 도 2에 나타난 바와 같이, 유리당류 분획(A), 또는 장티푸스균의 피막 다당류 및 디프테리아 톡소이드 접합 단백질의 접합체 분획(B)에 포함되는 다당류의 피크가 잘 분리됨을 확인하였다. 유리당류의 함량은, 상기 분리된 피크를 이용하여 HPAEC-PAD 크로마토그램으로 정량을 한 뒤, 총 다당류 대비 유리당류의 함량으로 결정하였다. 그 결과, 장티푸스균의 피막 다당류-디프테리아 톡소이드 접합체의 유리당류 함량은 0 내지 20%로 확인되었다.
As a result, as shown in Figure 2, it was confirmed that the peak of the polysaccharide contained in the free saccharide fraction (A) or the conjugate fraction (B) of the capsular polysaccharide of typhoid bacteria and diphtheria toxoid conjugated protein was well separated. The content of free saccharides was quantified using the HPAEC-PAD chromatogram using the separated peaks, and then determined as the content of free saccharides relative to the total polysaccharides. As a result, it was confirmed that the free saccharide content of the polysaccharide-diphtheria toxoid conjugate of typhoid bacteria was 0 to 20%.
1-3. 유리당류 함량 확인 방법의 적합성 평가1-3. Evaluation of the suitability of the method for determining the free sugar content
장티푸스균의 피막 다당류, 디프테리아 톡소이드 접합 단백질, 상기 다당류 및 접합 단백질의 혼합물, 및 피막 다당류 및 접합 단백질의 접합체(conjugate)를 사용하여 HIC를 수행함으로써, 본 발명의 방법이 유리당류의 함량을 측정하는데 적합한지 여부를 확인하였다. 실험은 실험예 1-1과 동일한 방법으로 수행하였고, 그 결과를 도 3에, 소수성 수지와 각 시료와의 결합 정도(%)를 하기 표 2에 나타내었다.By performing HIC using a capsular polysaccharide of Typhoid bacteria, a diphtheria toxoid conjugated protein, a mixture of the polysaccharide and conjugated protein, and a conjugate of the capsular polysaccharide and conjugated protein, the method of the present invention is used to measure the content of free saccharides. It was checked whether it was suitable. The experiment was performed in the same manner as in Experimental Example 1-1, and the results are shown in Fig. 3, and the degree of binding (%) between the hydrophobic resin and each sample is shown in Table 2 below.
수지의 결합 정도(%)Capsular polysaccharides and
Resin bonding degree (%)
수지의 결합 정도(%)Conjugated proteins and
Resin bonding degree (%)
단백질 접합체Typhoid bacteria capsular polysaccharide-
Protein conjugate
도 3 및 표 2에 나타난 바와 같이, 장티푸스균의 피막 다당류는 수지에 결합하지 않고 용출되었고, 디프테리아 톡소이드 접합 단백질과 상기 다당류 및 접합 단백질의 접합체는 수지에 결합된 뒤 용출되었다.As shown in FIG. 3 and Table 2, the polysaccharide of typhoid bacteria was eluted without binding to the resin, and the diphtheria toxoid conjugated protein and the conjugate of the polysaccharide and the conjugated protein were eluted after binding to the resin.
이를 통해, HIC가 장티푸스 접합 백신으로부터 유리당류를 분리하는데 적합한 방법임을 확인하였다.
Through this, it was confirmed that HIC is a suitable method for separating free sugars from typhoid conjugated vaccines.
실험예Experimental example
2. 2.
수막구균의Meningococcal
활성 다당류-디프테리아 Active polysaccharide-diphtheria
톡소이드Toxoid
CRM197CRM197
접합체의 유리당류 함량 측정 Measurement of free sugar content of conjugate
2-1. 접합체에 포함된 유리당류의 분리2-1. Separation of free sugars contained in conjugates
실시예 2에서 제조한 수막구균 활성 다당류-디프테리아 톡소이드 CRM197 접합체를 장티푸스균 피막 다당류-단백질 접합체 대신 사용하고, 1 M의 황산암모늄을 사용한 것을 제외하고는, 상기 실험예 1-1과 동일한 조건 및 방법으로 유리당류 분획 및 접합체 분획을 수득하였다.
The same conditions and methods as in Experimental Example 1-1, except that the meningococcal active polysaccharide-diphtheria toxoid CRM197 conjugate prepared in Example 2 was used instead of the typhoid capsular polysaccharide-protein conjugate, and 1 M ammonium sulfate was used. As a result, a free sugar fraction and a conjugate fraction were obtained.
2-2. 유리당류의 함량 측정2-2. Measurement of free sugar content
실험예 2-1에서 분리한 유리당류를 사용하여 하기 방법으로 유리당류의 함량을 측정하였다.Using the free sugar isolated in Experimental Example 2-1, the content of free sugar was measured by the following method.
구체적으로, 실험예 2-1에서 수득한 접합체 분획에 트리플루오로 아세트산 및 아세트산이 최종 농도가 각각 2 N이 되도록 첨가하고, 혈청그룹 A, W135 및 Y 다당류는 100℃에서 3시간, 혈청그룹 C 다당류는 80℃에서 3시간 동안 반응시켰다. 가수분해된 시료는 진공 오븐에서 16 내지 24시간 동안 완전히 건조시킨 후, 건조된 시료에 0.5 ㎖의 증류수를 첨가하여 용해시켰다. 상기 용해물을 0.2 ㎛의 필터로 여과하여 준비하였다. 이후의 과정은 상기 실험예 1-2와 동일한 조건 및 방법으로 수행하였다.Specifically, trifluoroacetic acid and acetic acid were added to the conjugate fraction obtained in Experimental Example 2-1 so that the final concentration was 2 N, respectively, and serogroups A, W135 and Y polysaccharides were added at 100°C for 3 hours, serogroup C The polysaccharide was reacted at 80° C. for 3 hours. The hydrolyzed sample was completely dried in a vacuum oven for 16 to 24 hours, and then 0.5 ml of distilled water was added to the dried sample to dissolve. The lysate was prepared by filtering through a 0.2 μm filter. The following process was performed under the same conditions and methods as in Experimental Example 1-2.
그 결과, 수막구균의 활성 다당류-디프테리아 톡소이드 CRM197 접합체의 유리당류 함량은, 혈청그룹 A는 0 내지 10%, C는 2 내지 40%, W135는 20 내지 50% 및 Y는 20 내지 50%로 확인되었다.As a result, the content of free saccharides in the active polysaccharide-diphtheria toxoid CRM197 conjugate of meningococcal, serogroup A is 0-10%, C is 2-40%, W135 is 20-50%, and Y is 20-50%. Became.
따라서, 본 발명에 따른 방법은, 제조된 접합 백신에 포함되는 유리당류의 함량을 측정함으로써, 백신의 품질 평가에 사용될 수 있다.
Therefore, the method according to the present invention can be used for evaluating the quality of a vaccine by measuring the content of free saccharides contained in the prepared conjugated vaccine.
2-3. 유리당류 함량 확인 방법의 적합성 평가2-3. Evaluation of the suitability of the method for determining the free sugar content
수막구균의 피막 다당류, 디프테리아 톡소이드 CRM197 접합 단백질, 상기 다당류 및 접합 단백질의 혼합물, 및 피막 다당류 및 접합 단백질의 접합체(conjugate)를 사용하여 본 발명의 방법이 유리당류의 함량을 측정하는데 적합한지 여부를 확인하였다. 실험은 실험예 1-1과 동일한 방법으로 수행하였고, 그 결과, 소수성 수지와 각 시료와의 결합 정도(%)를 하기 표 3에 나타내었다.Capsular polysaccharide of meningococcal, diphtheria toxoid CRM197 conjugated protein, a mixture of the polysaccharide and conjugated protein, and conjugates of the capsular polysaccharide and conjugated protein are used to determine whether the method of the present invention is suitable for measuring the content of free saccharides. Confirmed. The experiment was performed in the same manner as in Experimental Example 1-1, and as a result, the degree of binding (%) between the hydrophobic resin and each sample is shown in Table 3 below.
수지의 결합 정도(%) Capsular polysaccharides and
Resin bonding degree (%)
수지의 결합 정도(%)Conjugated proteins and
Resin bonding degree (%)
혈청그룹 C: 8
혈청그룹 W135: 0
혈청그룹 Y: 6Serogroup A: 4
Serogroup C: 8
Serogroup W135: 0
Serogroup Y: 6
혈청그룹 C: 5
혈청그룹 W135: 3
혈청그룹 Y: 4Serogroup A: 2
Serogroup C: 5
Serogroup W135: 3
Serogroup Y: 4
표 3에 나타난 바와 같이, 상기 결과와 동일하게 수막구균 피막 다당류는 수지에 결합하지 않았고, 디프테리아 톡소이드 CRM197 접합 단백질과 상기 다당류 및 접합 단백질의 접합체는 수지에 결합된 뒤 용출되었다.As shown in Table 3, in the same manner as in the above results, the meningococcal capsular polysaccharide did not bind to the resin, and the diphtheria toxoid CRM197 conjugated protein and the conjugate of the polysaccharide and the conjugated protein were eluted after binding to the resin.
이를 통해, HIC가 수막구균 접합 백신으로부터 유리당류를 분리하는데 적합한 방법임을 확인하였다.Through this, it was confirmed that HIC is a suitable method for separating free saccharides from meningococcal conjugated vaccines.
Claims (17)
2) 소수성이 부여된 접합체로부터 유리당류를 분리하는 단계; 및
3) 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법으로서,
상기 소수성 부여가 황산암모늄의 첨가에 의한 것이며,
상기 황산암모늄이 0.7 내지 1.8 M의 농도로 첨가되고,
상기 유리당류의 함량은 HPAEC-PAD(high performance anion exchange chromatography pulsed with amperometric detection)를 사용하여 측정되는 것인, 품질 평가 방법.
1) imparting hydrophobicity to the capsular polysaccharide-conjugated protein conjugate;
2) separating the free saccharide from the conjugate to which hydrophobicity is imparted; And
3) A method for evaluating the quality of a capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide,
The hydrophobic imparting is due to the addition of ammonium sulfate,
The ammonium sulfate is added at a concentration of 0.7 to 1.8 M,
The content of the free sugar is measured using HPAEC-PAD (high performance anion exchange chromatography pulsed with amperometric detection), the quality evaluation method.
The quality evaluation method according to claim 1, wherein the film polysaccharide is a bacterial film polysaccharide.
The method of claim 2, wherein the bacterial capsular polysaccharide is Streptococcus pneumoniae capsular polysaccharide, Neisseria meningitidis ) capsular polysaccharides, Salmonella typhi capsular polysaccharides, Haemophilus influenza B type capsular polysaccharides, and a quality evaluation method selected from the group consisting of a combination thereof.
The quality evaluation method according to claim 3, wherein the bacterial coating polysaccharide is a typhoid bacteria coating polysaccharide.
The quality evaluation method according to claim 3, wherein the bacterial capsular polysaccharide is a meningococcal capsular polysaccharide.
The method of claim 5, wherein the meningococcal capsular polysaccharide is serogroup A, C, W135 or Y.
The method of claim 1, wherein the conjugated protein is selected from the group consisting of diphtheria toxoid, diphtheria toxoid detoxifying variant CRM197, tetanus toxoid, and combinations thereof.
The quality evaluation method according to claim 7, wherein the conjugated protein is diphtheria toxoid.
The method according to claim 7, wherein the conjugated protein is a diphtheria toxoid detoxified variant CRM197.
The method of claim 1, wherein the separation is performed using hydrophobic interaction chromatography or an aluminum hydroxide adsorption method.
The quality evaluation method according to claim 13, wherein the hydrophobic interaction chromatography uses a resin having a ligand selected from the group consisting of phenyl, octyl, butyl and combinations thereof as column filler.
The method of claim 13, wherein the concentration of the conjugate applied to the resin in the hydrophobic interaction chromatography is 0.2 to 2.0 mg/ml.
2) 소수성이 부여된 접합 백신으로부터 유리당류를 분리하는 단계; 및
3) 분리된 유리당류의 함량을 측정하는 단계를 포함하는, 피막 다당류-단백질 접합 백신의 품질 평가 방법으로서,
상기 소수성 부여가 황산암모늄의 첨가에 의한 것이며,
상기 황산암모늄이 0.7 내지 1.8 M의 농도로 첨가되고,
상기 유리당류의 함량은 HPAEC-PAD(high performance anion exchange chromatography pulsed with amperometric detection)를 사용하여 측정되는 것인, 품질 평가 방법.1) imparting hydrophobicity to the conjugated vaccine of the capsular polysaccharide-conjugated protein;
2) separating the free saccharide from the conjugated vaccine imparted with hydrophobicity; And
3) A method for evaluating the quality of a capsular polysaccharide-protein conjugated vaccine comprising the step of measuring the content of the separated free saccharide,
The imparting of hydrophobicity is due to the addition of ammonium sulfate,
The ammonium sulfate is added at a concentration of 0.7 to 1.8 M,
The content of the free sugar is measured using HPAEC-PAD (high performance anion exchange chromatography pulsed with amperometric detection), the quality evaluation method.
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