WO2022114735A1 - Quality evaluation method for process of purifying prp antigen of haemophilus influenzae type b - Google Patents

Quality evaluation method for process of purifying prp antigen of haemophilus influenzae type b Download PDF

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WO2022114735A1
WO2022114735A1 PCT/KR2021/017295 KR2021017295W WO2022114735A1 WO 2022114735 A1 WO2022114735 A1 WO 2022114735A1 KR 2021017295 W KR2021017295 W KR 2021017295W WO 2022114735 A1 WO2022114735 A1 WO 2022114735A1
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prp
molecular weight
raw
hib
crude
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PCT/KR2021/017295
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French (fr)
Korean (ko)
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안교은
여동준
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주식회사 엘지화학
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/50—Organo-phosphines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/50—Organo-phosphines
    • C07F9/5004—Acyclic saturated phosphines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/89—Inverse chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
    • G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds

Definitions

  • the present invention relates to a quality evaluation method of a polyribosyl ribitol phosphate (PRP) antigen purification process of Haemophilus influenzae type b (Hib). More specifically, the present invention relates to a quality evaluation method in the purification process of polyribosyl ribitol phosphate (PRP), which is a raw material for the production of a Haemophilus influenzae type b vaccine.
  • PRP polyribosyl ribitol phosphate
  • Haemophilus influenzae type b (Haemophilus Influenza Type b, Hib) is a major cause of invasive and local bacterial infections such as meningitis, bacteremia, epiglottis, pneumonia, and suppurative arthritis in children.
  • Polyribosyl ribitol phosphate (PRP) of Haemophilus influenzae type b (Hib) is a major polysaccharide component constituting the coating of Hib.
  • the capsule polysaccharide capsule of Haemophilus influenzae type b (Hib) is a major virulence factor.
  • Antibodies to PRP are a major contributor to the sterilization and inhibition of infected Hib in serum.
  • PRP alone is used not only as a vaccine component, but also as a conjugate vaccine in which PRP is covalently linked to a T-cell-dependent protein antigen.
  • Hib's PRP is produced in the final raw form by culturing Haemophilus influenzae type b and purifying it.
  • IPC In Process Control
  • the present inventors tried to develop an IPC (In Process Control) method capable of pre-evaluating and predicting the quality of the Hib PRP product finally produced in the process stage of manufacturing Hib PRP.
  • IPC In Process Control
  • an object of the present invention is to provide a method for evaluating the quality of the final raw PRP in the process of manufacturing the raw PRP by purifying the PRP of Hib.
  • One aspect of the present invention provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
  • step (d) using the molecular weight data of the crude PRP measured in step (b) and the molecular weight distribution data of the crude PRP measured in step (c), a regression analysis between the molecular weight of the crude PRP and the molecular weight distribution of the crude PRP was performed performing;
  • Another aspect of the present invention provides a method for evaluating the quality of PRP in a process for purifying PRP of Hib, comprising the steps of:
  • step (b') using the molecular weight data of the raw PRP and the molecular weight distribution data of the raw PRP measured in step (a'), performing a regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution;
  • One aspect of the present invention provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
  • step (d) using the molecular weight data of the crude PRP measured in step (b) and the molecular weight distribution data of the crude PRP measured in step (c), a regression analysis between the molecular weight of the crude PRP and the molecular weight distribution of the crude PRP was performed performing;
  • the collection of a sample containing the crude PRP of Hib in step (a) of the present invention may be performed in an intermediate step of the purification process for purifying PRP from a culture in which Hib is cultured.
  • the term "crude PRP" refers to PRP that has not been purified as an intermediate product in the PRP purification process.
  • the process of purifying the PRP of Hib may include a process of treating ethanol (EtOH) or a process of treating diatomaceous earth.
  • the determination of the molecular weight of the crude PRP in the sample taken in step (b) may be performed using a chromatography method on a sample comprising crude PRP.
  • the chromatography method may be a liquid chromatography method using a column, for example, ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography method,
  • the present invention is not limited thereto.
  • the measurement of the molecular weight of the crude PRP in the sample taken in step (b) of the present invention may be performed by further comprising a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light Scattering (Multi-Angle Light Scattering, MALS) detection method may be further included.
  • a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light Scattering (Multi-Angle Light Scattering, MALS) detection method may be further included.
  • MALS Multi-Angle Light Scattering
  • the measurement of the molecular weight of the crude PRP in the sample taken in step (b) of the present invention can be performed by combining the above-described chromatography method with an ultraviolet absorbance detection method or a light scattering detection method, specifically, the reverse phase A chromatographic method and a multi-angle light scattering detection method can be combined.
  • step (c) the molecular weight distribution of raw PRP is measured from the final purified PRP.
  • raw PRP refers to PRP contained in the final purified raw material.
  • molecular weight distribution refers to a degree or shape in which molecular weights of polymers having various molecular weights are distributed.
  • the molecular weight distribution may be expressed as a number distribution curve, a weight distribution curve, or a cumulative distribution curve, and may also be expressed as a poly dispersity index (PDI).
  • PDI poly dispersity index
  • the molecular weight distribution measurement of the raw PRP of the present invention may be performed using a chromatography analysis method for a sample containing the raw PRP.
  • the chromatography analysis method may be, for example, size exclusion chromatography or gel permeation chromatography.
  • one or more standard substances with known molecular weight may be used.
  • the crude PRP molecular weight and the raw PRP molecular weight distribution using the molecular weight data of the crude PRP measured in the step (b) and the molecular weight distribution data of the raw PRP measured in the step (c) are used.
  • a regression analysis of the liver is performed.
  • the regression analysis determines the molecular weight of the crude PRP measured in step (b) and the molecular weight distribution of the raw PRP measured in step (c) as each variable, and a linear regression between the two variables. regression) analysis.
  • the regression analysis may be performed through a least square method.
  • step (e) the crude PRP molecular weight that satisfies the molecular weight distribution of the raw PRP above a certain level is calculated from the regression analysis.
  • a value above a specific level of molecular weight distribution of the raw PRP may be set to an appropriate value according to the guidelines for product quality control of the final purified PRP, and is not limited to any specific value.
  • the retention time, partition coefficient, and size-exclusion chromatography of the raw PRP of the two standard substances fraction by size can be analyzed using the peak data.
  • the molecular weight distribution value of the raw PRP may be set to a value of 50% or more.
  • the molecular weight value of the crude PRP satisfying the molecular weight distribution of the raw PRP above the specific level may be calculated by the above-described regression equation having the molecular weight distribution of the raw PRP and the crude PRP molecular weight as variables.
  • the method may further include comparing the molecular weight value of crude PRP obtained in the purification process of Hib PRP with the molecular weight value of crude PRP calculated in step (e). have.
  • the calculated molecular weight of the crude PRP that satisfies the molecular weight distribution of the raw PRP above the specific level can be utilized for IPC (In Process Control) as follows: Produce raw PRP having a molecular weight distribution above a specific level in the PRP raw material In order to do this, the molecular weight of crude PRP in the sample taken at the intermediate stage of the PRP purification process must have the value of crude PRP molecular weight calculated by the regression analysis. Therefore, by monitoring whether the molecular weight of the crude PRP in the sample collected during the PRP purification process has the calculated crude PRP molecular weight value, it can be utilized for IPC (In Process Control) of PRP production.
  • IPC In Process Control
  • Another aspect of the present application provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
  • step (b') using the molecular weight data of the raw PRP and the molecular weight distribution data of the raw PRP measured in step (a'), performing a regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution;
  • step (a') the molecular weight of the raw PRP is measured.
  • the measurement of the molecular weight of crude PRP in step (a') may be performed on a sample containing crude PRP through a chromatography method, and the chromatography method is ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography method.
  • the measurement of the molecular weight of raw PRP in step (a') of the present invention may be performed by further including a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light scattering detection (Multi-Angle Light Scattering, MALS) method can be further included.
  • a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light scattering detection (Multi-Angle Light Scattering, MALS) method can be further included.
  • MALS Multi-Angle Light Scattering
  • the measurement of the molecular weight of raw PRP in step (a') of the present invention may be performed by combining the above-described chromatography method with an ultraviolet absorbance detection method or a multi-angle light scattering detection method, specifically, reverse phase chromatography It can be carried out by combining a method for detecting a lithography method and a method for detecting multi-angle light scattering.
  • step (a') the molecular weight distribution of the raw PRP is measured.
  • the molecular weight distribution measurement of the raw PRP of the present invention may be performed using a chromatography analysis method for a sample containing the raw PRP.
  • the chromatography method may be, for example, size exclusion chromatography or gel permeation chromatography.
  • one or more standard substances with known molecular weight may be used.
  • step (b') using the molecular weight data of the raw PRP measured in step (a') and the molecular weight distribution data of the raw PRP, regression between the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP analysis) is performed.
  • the regression analysis is to determine the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP measured in step (a') as each variable, and perform a linear regression analysis between the two variables.
  • the regression analysis may be performed through a least square method. Through the regression analysis, a regression equation having the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP as variables can be derived.
  • step (c') the molecular weight of the raw PRP that satisfies the molecular weight distribution of the raw PRP above a certain level is calculated from the regression analysis.
  • step (c') the value above a specific level of molecular weight distribution of the raw PRP may be set to an appropriate value according to the guidelines for product quality control of the final purified PRP, and is not limited to any specific value.
  • the retention time, partition coefficient, and size-exclusion chromatography of the raw PRP of the two standard substances fraction by size can be analyzed using the peak data.
  • the molecular weight distribution value of the raw PRP may be set to a value of 50% or more.
  • the molecular weight of the raw PRP that satisfies the molecular weight distribution of the raw PRP above the specific level can be calculated by the above-described regression equation having the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP as variables.
  • the method further comprises comparing the molecular weight value of raw PRP obtained in the purification process of Hib PRP with the molecular weight value of raw PRP calculated in step (c'). can do.
  • the calculated molecular weight of the raw PRP which satisfies the molecular weight distribution of the raw PRP above the specific level, can be utilized for IPC (In Process Control) as follows: Produce raw PRP having a molecular weight distribution above a specific level in the PRP raw material In order to do this, the molecular weight of the raw PRP in the raw PRP purification final product must have the raw PRP molecular weight value calculated by the regression analysis. Therefore, by monitoring whether the molecular weight of raw PRP in the final product of PRP purification has the calculated raw PRP molecular weight value, it can be utilized for IPC (In Process Control) of PRP production.
  • IPC In Process Control
  • the molecular weight value of crude PRP and the molecular weight value of raw PRP calculated according to the method of the present invention described above may be used alone or in combination with each other in the method for evaluating the quality of PRP of the present invention.
  • the PRP quality evaluation method of the present invention by measuring the molecular weight value of crude PRP or the molecular weight value of raw PRP through regression analysis in the PRP purification process step of Hib, the quality of the Hib PRP product finally produced can be evaluated and predicted in advance.
  • the method of the present invention can be applied as an IPC (In Process Control) method in the intermediate stage of the purification process of Hib PRP raw powder, and can be usefully utilized to confirm the timing of quality issues occurring in the actual process.
  • IPC In Process Control
  • FIG. 3 is a schematic diagram showing the concept of performing IPC (In Process Control) in the PRP production process of Hib of the present invention.
  • the collected sample containing crude PRP is put into a reversed-phase chromatography column, and the molecular weight of crude PRP is measured using a MALS (Multi-Angle Light Scattering, MALS) detector for the crude PRP that has passed through without binding to the column.
  • MALS Multi-Angle Light Scattering, MALS
  • PRP was purified from the culture medium of Haemophilus influenzae type b (Hib), and the molecular weight distribution of raw PRP was measured in the final purified PRP raw material.
  • the molecular weight distribution of raw PRP was measured through the following procedure.
  • the molecular weight of crude PRP must be 38.5 kDa or more in order to produce raw PRP having a molecular weight distribution of 50% or more of raw PRP. Based on the above analysis, if the molecular weight of crude PRP is managed to be 38.5 kDa or more during the purification process of PRP, the molecular weight distribution of raw PRP, which is a quality control standard, is 50% or more, the final purified PRP raw powder can get
  • PRP was purified from the culture medium of Haemophilus influenzae type b (Hib) to prepare the final purified PRP raw material.
  • the molecular weight of the raw PRP was measured.
  • the molecular weight measurement of raw PRP was performed in the same manner as the crude PRP molecular weight measurement of Experimental Example 1-1.
  • a sample containing raw PRP was put into a reverse phase chromatography column, and the molecular weight of raw PRP was measured using a MALS (Multi-Angle Light Scattering, MALS) detector for raw PRP that passed through without binding to the column.
  • MALS Multi-Angle Light Scattering, MALS
  • PRP was purified from the culture medium of Haemophilus influenzae type b (Hib), and the molecular weight distribution of the raw PRP was measured in the final purified PRP raw material. The molecular weight distribution of raw PRP was measured in the same manner as described in Experimental Example 1-2.
  • x represents the molecular weight of the raw PRP
  • y represents the molecular weight distribution of the raw PRP
  • the R square value (R 2 ) representing the reliability (explanatory power) of the regression equation was 0.9222. It was confirmed that the R-squared value (R 2 ) was 0.9222, indicating a high correlation. It can be seen from the regression equation that the molecular weight of raw PRP must be 69.1 kDa or more in order to produce raw PRP having a molecular weight distribution of 50% or more of raw PRP.
  • the molecular weight of raw PRP is managed to be 69.1 kDa or more during the purification process of PRP, the molecular weight distribution of raw PRP, which is a quality control standard, is 50% or more, the final purified PRP raw material can get

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Abstract

The present invention relates to a quality evaluation method for a process of purifying a PRP antigen of Haemophilus influenzae type b (Hib). According to the method of the present invention, the molecular weight of crude PRP or pure PRP is measured in a process of purifying the PRP of Hib, whereby the quality of the finally produced Hib PRP product can be evaluated and predicted in advance.

Description

헤모필루스 인플루엔자 타입 B의 PRP 항원 정제 공정의 품질 평가 방법 Method for quality evaluation of PRP antigen purification process of Haemophilus influenzae type B
관련출원과의 상호인용Cross Citation with Related Applications
본 출원은 2020년 11월 25일자 한국 특허 출원 제10-2020-0159439호에 기초한 우선권의 이익을 주장하며, 해당 한국 특허 출원의 문헌에 개시된 모든 내용은 본 명세서의 일부로서 포함된다. This application claims the benefit of priority based on Korean Patent Application No. 10-2020-0159439 dated November 25, 2020, and all contents disclosed in the literature of the Korean patent application are incorporated as a part of this specification.
기술분야technical field
본 발명은 헤모필루스 인플루엔자 타입 b(Hib)의 폴리리보실 리비톨 포스페이트(polyribosyl ribitol phosphate, PRP) 항원 정제 공정의 품질 평가 방법에 관한 것이다. 보다 구체적으로는, 본 발명은 헤모필루스 인플루엔자 타입 b의 백신 제조의 원료가 되는 폴리리보실 리비톨 포스페이트(PRP)의 정제 공정에서의 품질 평가 방법에 관한 것이다. The present invention relates to a quality evaluation method of a polyribosyl ribitol phosphate (PRP) antigen purification process of Haemophilus influenzae type b (Hib). More specifically, the present invention relates to a quality evaluation method in the purification process of polyribosyl ribitol phosphate (PRP), which is a raw material for the production of a Haemophilus influenzae type b vaccine.
헤모필루스 인플루엔자 타입 b (Haemophilus Influenza Type b, Hib)는 소아에게서 수막염, 균혈증, 후두개염, 폐렴, 화농성 관절염 등 침습성 및 국소성 세균 감염을 일으키는 주요 원인균이다. 헤모필루스 인플루엔자 타입 b(Hib)의 폴리리보실 리비톨 포스페이트(polyribosyl ribitol phosphate, PRP)는 Hib의 피막을 구성하는 주요 다당류 성분이다. 헤모필루스 인플루엔자 타입 b(Hib)의 피막의 다당류 캡슐은 주요한 독성 인자(virulence factor)가 된다. Haemophilus influenzae type b (Haemophilus Influenza Type b, Hib) is a major cause of invasive and local bacterial infections such as meningitis, bacteremia, epiglottis, pneumonia, and suppurative arthritis in children. Polyribosyl ribitol phosphate (PRP) of Haemophilus influenzae type b (Hib) is a major polysaccharide component constituting the coating of Hib. The capsule polysaccharide capsule of Haemophilus influenzae type b (Hib) is a major virulence factor.
PRP에 대한 항체는 혈청에서 감염된 Hib를 살균하여 억제하는데 주요한 기여자가 된다. PRP 단독으로도 백신 성분으로 이용될 뿐만 아니라, PRP를 T-세포 의존적 단백질 항원과 공유결합시킨 접합 백신으로도 이용되고 있다. Antibodies to PRP are a major contributor to the sterilization and inhibition of infected Hib in serum. PRP alone is used not only as a vaccine component, but also as a conjugate vaccine in which PRP is covalently linked to a T-cell-dependent protein antigen.
Hib의 PRP는 헤모필루스 인플루엔자 타입 b를 배양하여 이를 정제하는 공정을 거쳐 최종 원말 형태로 제조하고 있으나, 최종 제품에서 품질 이슈가 발생한 경우 생산된 배치(batch)를 전량 폐기해야 하므로 비용상 손실이 크다는 문제가 있다. 현재까지 Hib의 PRP를 정제하는 공정 단계에서, 최종적으로 생산되는 PRP 제품의 품질을 미리 평가하고 예측할 수 있는 IPC (In Process Control)방법이 개발되어 있지 않다. Hib's PRP is produced in the final raw form by culturing Haemophilus influenzae type b and purifying it. there is To date, in the process step of refining PRP of Hib, an IPC (In Process Control) method that can pre-evaluate and predict the quality of the finally produced PRP product has not been developed.
[선행기술문헌] [Prior art literature]
[특허문헌] [Patent Literature]
미국등록특허 제4,220,717호 US Patent No. 4,220,717
본 발명자들은 Hib의 PRP를 제조하는 공정 단계에서, 최종적으로 생산되는 Hib의 PRP 제품의 품질을 미리 평가하고 예측할 수 있는 IPC (In Process Control) 방법을 개발하기 위해 연구 노력하였다. 그 결과, Hib의 PRP 정제 공정에서 중간 산물인 조(crude) PRP 분자량 데이터 또는 최종 산물인 원말의 원말 PRP 분자량 데이터와, 최종 산물의 원말 PRP의 분자량 분포 데이터를 이용한 회귀 분석을 통해, 정제되는 PRP의 품질을 미리 평가하고 예측할 수 있음을 증명하여 본 발명을 완성하였다. The present inventors tried to develop an IPC (In Process Control) method capable of pre-evaluating and predicting the quality of the Hib PRP product finally produced in the process stage of manufacturing Hib PRP. As a result, in the PRP purification process of Hib, crude PRP molecular weight data, an intermediate product, or raw PRP molecular weight data of the final product, and molecular weight distribution data of raw PRP of the final product. PRP purified through regression analysis The present invention was completed by proving that it is possible to evaluate and predict the quality in advance.
따라서, 본 발명의 목적은 Hib의 PRP를 정제하여 원말 PRP를 제조하는 공정에서 최종 원말 PRP의 품질을 평가하는 방법을 제공하는 것에 있다. Accordingly, an object of the present invention is to provide a method for evaluating the quality of the final raw PRP in the process of manufacturing the raw PRP by purifying the PRP of Hib.
상기한 본 발명의 목적을 달성하기 위해, In order to achieve the above object of the present invention,
본 발명의 일 측면은, 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법을 제공한다: One aspect of the present invention provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
(a) 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하는 공정 중에서 조(crude) PRP를 포함하는 샘플을 채취하는 단계; (a) Collecting a sample containing crude PRP in the process of purifying polyribosyl ribitol phosphate (PRP) from a culture solution of Haemophilus influenzae type b (Hib) step;
(b) 채취한 샘플 내의 조 PRP의 분자량을 측정하는 단계; (b) measuring the molecular weight of the crude PRP in the collected sample;
(c) PRP를 최종 정제한 원말에서 원말 PRP의 분자량 분포를 측정하는 단계; (c) measuring the molecular weight distribution of the raw PRP in the final purified raw PRP;
(d) 단계 (b)에서 측정한 조 PRP의 분자량 데이터와, 단계 (c)에서 측정한 원말 PRP의 분자량 분포 데이터를 사용하여, 조 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (d) using the molecular weight data of the crude PRP measured in step (b) and the molecular weight distribution data of the crude PRP measured in step (c), a regression analysis between the molecular weight of the crude PRP and the molecular weight distribution of the crude PRP was performed performing; and
(e) 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 조 PRP 분자량을 산출하는 단계. (e) calculating a crude PRP molecular weight that satisfies the original PRP molecular weight distribution above a certain level from the regression analysis.
본 발명의 다른 측면은, 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법을 제공한다: Another aspect of the present invention provides a method for evaluating the quality of PRP in a process for purifying PRP of Hib, comprising the steps of:
(a') 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하여 얻은 최종 정제 원말에서, 원말 PRP의 분자량 및 분자량 분포를 측정하는 단계; (a') In the final purified raw material obtained by purifying polyribosyl ribitol phosphate (PRP) from the culture medium of Haemophilus influenzae type b (Hib), the molecular weight and molecular weight distribution of the raw PRP measuring;
(b') 단계 (a')에서 측정한 원말 PRP의 분자량 데이터와 원말 PRP의 분자량 분포 데이터를 사용하여, 원말 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (b') using the molecular weight data of the raw PRP and the molecular weight distribution data of the raw PRP measured in step (a'), performing a regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution; and
(c') 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 원말 PRP 분자량을 산출하는 단계. (c ') calculating the raw PRP molecular weight that satisfies the raw PRP molecular weight distribution above a specific level from the regression analysis.
이하, 본 발명에 대한 이해를 돕기 위해 본 발명을 더욱 상세하게 설명한다. 이때, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니 되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다.Hereinafter, the present invention will be described in more detail to help the understanding of the present invention. At this time, the terms or words used in the present specification and claims should not be construed as being limited to conventional or dictionary meanings, and the inventor appropriately defines the concept of the term in order to describe his invention in the best way. Based on the principle that it can be done, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.
본 발명의 일 측면은 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법을 제공한다: One aspect of the present invention provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
(a) 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하는 공정 중에서 조(crude) PRP를 포함하는 샘플을 채취하는 단계; (a) Collecting a sample containing crude PRP in the process of purifying polyribosyl ribitol phosphate (PRP) from a culture solution of Haemophilus influenzae type b (Hib) step;
(b) 채취한 샘플 내의 조 PRP의 분자량을 측정하는 단계; (b) measuring the molecular weight of the crude PRP in the collected sample;
(c) PRP를 최종 정제한 원말에서 원말 PRP의 분자량 분포를 측정하는 단계; (c) measuring the molecular weight distribution of the raw PRP in the final purified raw PRP;
(d) 단계 (b)에서 측정한 조 PRP의 분자량 데이터와, 단계 (c)에서 측정한 원말 PRP의 분자량 분포 데이터를 사용하여, 조 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (d) using the molecular weight data of the crude PRP measured in step (b) and the molecular weight distribution data of the crude PRP measured in step (c), a regression analysis between the molecular weight of the crude PRP and the molecular weight distribution of the crude PRP was performed performing; and
(e) 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 조 PRP 분자량을 산출하는 단계. (e) calculating a crude PRP molecular weight that satisfies the original PRP molecular weight distribution above a certain level from the regression analysis.
본 발명의 상기 단계 (a)의 Hib의 조 PRP를 포함하는 샘플의 채취는 Hib를 배양한 배양물로부터 PRP를 정제하는 정제 공정 중간 단계에서 이루어질 수 있다. 상기 용어 "조 PRP"는 PRP 정제 공정 중의 중간산물로 정제가 완료되지 않은 PRP를 의미한다. 상기 Hib의 PRP를 정제하는 공정은 에탄올(EtOH)를 처리하는 공정, 또는 규조토를 처리하는 공정을 포함할 수 있다. The collection of a sample containing the crude PRP of Hib in step (a) of the present invention may be performed in an intermediate step of the purification process for purifying PRP from a culture in which Hib is cultured. The term "crude PRP" refers to PRP that has not been purified as an intermediate product in the PRP purification process. The process of purifying the PRP of Hib may include a process of treating ethanol (EtOH) or a process of treating diatomaceous earth.
일 구현예에서, 단계 (b)에서 채취한 샘플내의 조 PRP의 분자량의 측정은 조 PRP를 포함하는 샘플에 대해 크로마토그래피(chromatography) 방법을 사용하여 행할 수 있다. 상기 크로마토그래피 방법은 컬럼을 사용하는 액체 크로마토그래피 방법일 수 있으며, 예를 들어 이온교환 크로마토그래피, 친화성 크로마토그래피, 크기배제 크로마토그래피, 역상 크로마토그래피, 또는 소수성 상호작용 크로마토그래피 방법일 수 있으나, 이에 한정되지 않는다. In one embodiment, the determination of the molecular weight of the crude PRP in the sample taken in step (b) may be performed using a chromatography method on a sample comprising crude PRP. The chromatography method may be a liquid chromatography method using a column, for example, ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography method, However, the present invention is not limited thereto.
다른 구현예에서, 본 발명의 단계 (b)에서 채취한 샘플내의 조 PRP의 분자량의 측정은 크로마토그래피 방법에 더해, 자외선 흡광도 검출 방법 또는 광산란 검출 방법을 더 포함하여 행할 수 있으며, 구체적으로 다각도 광 산란(Multi-Angle Light Scattering, MALS) 검출 방법을 더 포함하여 행할 수 있다. In another embodiment, the measurement of the molecular weight of the crude PRP in the sample taken in step (b) of the present invention may be performed by further comprising a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light Scattering (Multi-Angle Light Scattering, MALS) detection method may be further included.
또 다른 구현예에서, 본 발명의 단계 (b)에서 채취한 샘플내의 조 PRP의 분자량의 측정은 상술한 크로마토그래피 방법에 자외선 흡광도 검출 방법 또는 광산란 검출 방법을 결합하여 행할 수 있고, 구체적으로는 역상 크로마토그래피 방법 및 다각도 광 산란 검출 방법을 결합하여 행할 수 있다. In another embodiment, the measurement of the molecular weight of the crude PRP in the sample taken in step (b) of the present invention can be performed by combining the above-described chromatography method with an ultraviolet absorbance detection method or a light scattering detection method, specifically, the reverse phase A chromatographic method and a multi-angle light scattering detection method can be combined.
상기 단계 (c)에서 PRP를 최종 정제한 원말에서 원말 PRP의 분자량 분포를 측정한다. 상기 용어 "원말 PRP"는 최종 정제한 원말에 포함되어 있는 PRP를 의미한다. In step (c), the molecular weight distribution of raw PRP is measured from the final purified PRP. The term "raw PRP" refers to PRP contained in the final purified raw material.
본 명세서에서 사용되는 용어 "분자량 분포(molecular weight distribution)"는 다양한 분자량을 갖는 고분자의 분자량이 분포된 정도 또는 모양을 의미한다. 상기 분자량 분포는 수 분포곡선, 중량 분포곡선, 또는 누적 분포곡선으로 표현될 수 있으며, 다분산지수(PDI, poly dispersity index)으로도 표현될 수 있다. As used herein, the term “molecular weight distribution” refers to a degree or shape in which molecular weights of polymers having various molecular weights are distributed. The molecular weight distribution may be expressed as a number distribution curve, a weight distribution curve, or a cumulative distribution curve, and may also be expressed as a poly dispersity index (PDI).
일 구현예에서, 본 발명의 원말 PRP의 분자량 분포 측정은 원말 PRP를 포함하는 샘플에 대한 크로마토그래피 분석 방법을 이용하여 행할 수 있다. 상기 크로마토그래피 분석 방법은 예를 들어, 크기 배제 크로마토그래피(size exclusion chromatography) 또는 겔 침투 크로마토그래피(gel permeation chromatography)일 수 있다. 상기 크로마토그래피 방법을 이용한 원말 PRP의 분자량 분포 측정시에 분자량을 알고 있는 하나 이상의 표준 물질을 사용할 수 있다. In one embodiment, the molecular weight distribution measurement of the raw PRP of the present invention may be performed using a chromatography analysis method for a sample containing the raw PRP. The chromatography analysis method may be, for example, size exclusion chromatography or gel permeation chromatography. When measuring the molecular weight distribution of raw PRP using the chromatography method, one or more standard substances with known molecular weight may be used.
상기 단계 (d)에서는, 상기 단계 (b)에서 측정된 조 PRP의 분자량 데이터와, 상기 단계 (c)에서 측정한 원말 PRP의 분자량 분포 데이터를 사용하여, 상기 조 PRP 분자량과 상기 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행한다. In the step (d), the crude PRP molecular weight and the raw PRP molecular weight distribution using the molecular weight data of the crude PRP measured in the step (b) and the molecular weight distribution data of the raw PRP measured in the step (c) are used. A regression analysis of the liver is performed.
일 구현예에서, 상기 회귀 분석은 단계 (b)에서 측정된 조 PRP의 분자량과, 단계 (c)에서 측정한 원말 PRP의 분자량 분포를 각각의 변수로 정하고, 상기 두개의 변수간의 선형 회귀(linear regression) 분석을 행하는 것일 수 있다. In one embodiment, the regression analysis determines the molecular weight of the crude PRP measured in step (b) and the molecular weight distribution of the raw PRP measured in step (c) as each variable, and a linear regression between the two variables. regression) analysis.
일 구현예에서, 상기 회귀 분석은 최소제곱법(least square method)를 통해 수행할 수 있다. In an embodiment, the regression analysis may be performed through a least square method.
상기 회귀 분석을 통해 조 PRP의 분자량과 원말 PRP의 분자량 분포를 각각 변수로 갖는 회귀 방정식이 유도될 수 있다. Through the regression analysis, a regression equation having the molecular weight of crude PRP and the molecular weight distribution of raw PRP as variables can be derived.
상기 단계 (e)에서는 상기 회귀 분석으로부터 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는 조 PRP 분자량을 산출한다. In step (e), the crude PRP molecular weight that satisfies the molecular weight distribution of the raw PRP above a certain level is calculated from the regression analysis.
단계 (e)에서 상기 원말 PRP의 분자량 분포의 특정 수준 이상의 값은 최종 정제된 PRP의 제품 품질 관리(quality control) 지침에 따라 적절한 값으로 설정될 수 있으며, 어느 특정 값으로 한정되지 않는다. In step (e), a value above a specific level of molecular weight distribution of the raw PRP may be set to an appropriate value according to the guidelines for product quality control of the final purified PRP, and is not limited to any specific value.
구체적인 일 구현예에서, 2가지의 표준 물질과 크기배제 크로마토그래피를 이용한 원말 PRP 분자량 분포 분석에서, 상기 2가지 표준 물질의 머무름 시간(retension time), 분배계수 및 원말 PRP의 크기배제 크로마토그래피 크기별 분획 피크 데이터를 이용하여 원말 PRP의 분자량 분포를 분석할 수 있다. In a specific embodiment, in the molecular weight distribution analysis of raw PRP using two standard substances and size exclusion chromatography, the retention time, partition coefficient, and size-exclusion chromatography of the raw PRP of the two standard substances fraction by size The molecular weight distribution of raw PRP can be analyzed using the peak data.
구체적인 일 구현예에서, 상기 원말 PRP의 분자량 분포 값은 50% 이상의 값으로 설정될 수 있다. In a specific embodiment, the molecular weight distribution value of the raw PRP may be set to a value of 50% or more.
상기 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는 조 PRP의 분자량 값은, 원말 PRP의 분자량 분포와 조 PRP 분자량을 변수로 갖는 상술한 회귀 방정식에 의해 산출될 수 있다. The molecular weight value of the crude PRP satisfying the molecular weight distribution of the raw PRP above the specific level may be calculated by the above-described regression equation having the molecular weight distribution of the raw PRP and the crude PRP molecular weight as variables.
일 구현예에서, 상기 단계 (e)이후에, Hib의 PRP의 정제 공정에서 얻어지는 조 PRP의 분자량 값과, 상기 단계 (e)에서 산출된 조 PRP의 분자량 값을 비교하는 단계를 더 포함할 수 있다. In one embodiment, after step (e), the method may further include comparing the molecular weight value of crude PRP obtained in the purification process of Hib PRP with the molecular weight value of crude PRP calculated in step (e). have.
상기 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는, 상기 산출된 조 PRP의 분자량은 다음과 같이 IPC (In Process Control)에 활용될 수 있다: PRP 원말에서 특정 수준 이상의 분자량 분포를 갖는 원말 PRP를 생산하기 위해서는, PRP 정제 공정 중간 단계에서 채취한 샘플 중의 조 PRP의 분자량이, 상기 회귀 분석에 의해 산출된 조 PRP 분자량 값을 가져야 한다. 따라서, PRP 정제 공정 중에서 채취한 샘플 중의 조 PRP의 분자량이 상기 산출된 조 PRP 분자량 값을 갖는지를 모니터링하여 PRP 생산의 IPC(In Process Control)에 활용할 수 있다. The calculated molecular weight of the crude PRP that satisfies the molecular weight distribution of the raw PRP above the specific level can be utilized for IPC (In Process Control) as follows: Produce raw PRP having a molecular weight distribution above a specific level in the PRP raw material In order to do this, the molecular weight of crude PRP in the sample taken at the intermediate stage of the PRP purification process must have the value of crude PRP molecular weight calculated by the regression analysis. Therefore, by monitoring whether the molecular weight of the crude PRP in the sample collected during the PRP purification process has the calculated crude PRP molecular weight value, it can be utilized for IPC (In Process Control) of PRP production.
본 출원의 다른 측면은 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법을 제공한다: Another aspect of the present application provides a method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the following steps:
(a') 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하여 얻은 최종 정제 원말에서, 원말 PRP의 분자량 및 원말 PRP의 분자량 분포를 측정하는 단계; (a') In the final purified raw material obtained by purifying polyribosyl ribitol phosphate (PRP) from the culture medium of Haemophilus influenzae type b (Hib), the molecular weight of the raw PRP and the raw PRP measuring the molecular weight distribution of
(b') 단계 (a')에서 측정한 원말 PRP의 분자량 데이터와 원말 PRP의 분자량 분포 데이터를 사용하여, 원말 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (b') using the molecular weight data of the raw PRP and the molecular weight distribution data of the raw PRP measured in step (a'), performing a regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution; and
(c') 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 원말 PRP 분자량을 산출하는 단계. (c ') calculating the raw PRP molecular weight that satisfies the raw PRP molecular weight distribution above a specific level from the regression analysis.
상기 단계 (a')에서 원말 PRP의 분자량을 측정한다. In step (a'), the molecular weight of the raw PRP is measured.
일 구현예에서, 단계 (a')에서 원말 PRP의 분자량의 측정은 원말 PRP를 포함하는 샘플에 대해 크로마토그래피(chromatography) 방법을 통해 행할 수 있으며, 상기 크로마토그래피 방법은 이온교환 크로마토그래피, 친화성 크로마토그래피, 크기배제 크로마토그래피, 역상 크로마토그래피, 또는 소수성 상호작용 크로마토그래피 방법일 수 있다. In one embodiment, the measurement of the molecular weight of crude PRP in step (a') may be performed on a sample containing crude PRP through a chromatography method, and the chromatography method is ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography method.
다른 구현예에서, 본 발명의 단계 (a')에서 원말 PRP의 분자량의 측정은 상기 크로마토그래피 방법에 더해, 자외선 흡광도 검출 방법 또는 광산란 검출 방법을 더 포함하여 행할 수 있으며, 구체적으로 다각도 광 산란 검출(Multi-Angle Light Scattering, MALS) 방법을 더 포함하여 행할 수 있다. In another embodiment, the measurement of the molecular weight of raw PRP in step (a') of the present invention may be performed by further including a UV absorbance detection method or a light scattering detection method in addition to the chromatography method, specifically, multi-angle light scattering detection (Multi-Angle Light Scattering, MALS) method can be further included.
또 다른 구현예에서, 본 발명의 단계 (a')에서 원말 PRP의 분자량의 측정은 상술한 크로마토그래피 방법에, 자외선 흡광도 검출 방법 또는 다각도 광 산란 검출 방법을 결합하여 행할 수 있으며, 구체적으로 역상 크로마토그래피 방법 및 다각도 광 산란 검출 방법을 결합하여 행할 수 있다. In another embodiment, the measurement of the molecular weight of raw PRP in step (a') of the present invention may be performed by combining the above-described chromatography method with an ultraviolet absorbance detection method or a multi-angle light scattering detection method, specifically, reverse phase chromatography It can be carried out by combining a method for detecting a lithography method and a method for detecting multi-angle light scattering.
상기 단계 (a')에서 원말 PRP의 분자량 분포를 측정한다. In step (a'), the molecular weight distribution of the raw PRP is measured.
용어 "분자량 분포"에 대한 내용은 상술한 본 발명의 일 측면에서 설명된 것과 동일하므로, 중복하여 기재하지 않는다. Since the content of the term “molecular weight distribution” is the same as that described in the above-described aspect of the present invention, it will not be repeated.
일 구현예에서, 본 발명의 원말 PRP의 분자량 분포 측정은 원말 PRP를 포함하는 샘플에 대한 크로마토그래피 분석 방법을 이용하여 행할 수 있다. 상기 크로마토그래피 방법은 예를 들어, 크기 배제 크로마토그래피(size exclusion chromatography) 또는 겔 침투 크로마토그래피(gel permeation chromatography)일 수 있다. 상기 크로마토그래피 방법을 이용한 원말 PRP의 분자량 분포 측정시에 분자량을 알고 있는 하나 이상의 표준 물질을 사용할 수 있다. In one embodiment, the molecular weight distribution measurement of the raw PRP of the present invention may be performed using a chromatography analysis method for a sample containing the raw PRP. The chromatography method may be, for example, size exclusion chromatography or gel permeation chromatography. When measuring the molecular weight distribution of raw PRP using the chromatography method, one or more standard substances with known molecular weight may be used.
상기 단계 (b')에서는, 상기 단계 (a')에서 측정된 원말 PRP의 분자량 데이터와, 원말 PRP의 분자량 분포 데이터를 사용하여, 상기 원말 PRP의 분자량과 원말 PRP의 분자량 분포 간의 회귀 분석(regression analysis)을 수행한다. In the step (b'), using the molecular weight data of the raw PRP measured in step (a') and the molecular weight distribution data of the raw PRP, regression between the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP analysis) is performed.
일 구현예에서, 상기 회귀 분석은 단계 (a')에서 측정된 원말 PRP의 분자량과, 원말 PRP의 분자량 분포를 각각의 변수로 정하고, 상기 두개의 변수간의 선형 회귀(linear regression) 분석을 행하는 것일 수 있다. In one embodiment, the regression analysis is to determine the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP measured in step (a') as each variable, and perform a linear regression analysis between the two variables. can
일 구현예에서, 상기 회귀 분석은 최소제곱법(least square method)를 통해 수행할 수 있다. 상기 회귀 분석을 통해 원말 PRP의 분자량과 원말 PRP의 분자량 분포를 각각 변수로 갖는 회귀 방정식이 유도될 수 있다. In an embodiment, the regression analysis may be performed through a least square method. Through the regression analysis, a regression equation having the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP as variables can be derived.
상기 단계 (c')에서는 상기 회귀 분석으로부터 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는 원말 PRP의 분자량을 산출한다. In step (c'), the molecular weight of the raw PRP that satisfies the molecular weight distribution of the raw PRP above a certain level is calculated from the regression analysis.
단계 (c')에서 상기 원말 PRP의 분자량 분포의 특정 수준 이상의 값은 최종 정제된 PRP의 제품 품질 관리(quality control) 지침에 따라 적절한 값으로 설정될 수 있으며, 어느 특정 값으로 한정되지 않는다. In step (c'), the value above a specific level of molecular weight distribution of the raw PRP may be set to an appropriate value according to the guidelines for product quality control of the final purified PRP, and is not limited to any specific value.
구체적인 일 구현예에서, 2가지의 표준 물질과 크기배제 크로마토그래피를 이용한 원말 PRP 분자량 분포 분석에서, 상기 2가지 표준 물질의 머무름 시간(retension time), 분배계수 및 원말 PRP의 크기배제 크로마토그래피 크기별 분획 피크 데이터를 이용하여 원말 PRP의 분자량 분포를 분석할 수 있다. In a specific embodiment, in the molecular weight distribution analysis of raw PRP using two standard substances and size exclusion chromatography, the retention time, partition coefficient, and size-exclusion chromatography of the raw PRP of the two standard substances fraction by size The molecular weight distribution of raw PRP can be analyzed using the peak data.
구체적인 일 구현예에서, 상기 원말 PRP의 분자량 분포 값은 50% 이상의 값으로 설정될 수 있다. In a specific embodiment, the molecular weight distribution value of the raw PRP may be set to a value of 50% or more.
상기 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는 원말 PRP의 분자량은, 원말 PRP의 분자량과 원말 PRP의 분자량 분포를 변수로 갖는 상술한 회귀 방정식에 의해 산출될 수 있다. The molecular weight of the raw PRP that satisfies the molecular weight distribution of the raw PRP above the specific level can be calculated by the above-described regression equation having the molecular weight of the raw PRP and the molecular weight distribution of the raw PRP as variables.
일 구현예에서, 상기 단계 (c') 이후에, Hib의 PRP의 정제 공정에서 얻어지는 원말 PRP의 분자량 값과, 상기 단계 (c')에서 산출된 원말 PRP의 분자량 값을 비교하는 단계를 더 포함할 수 있다. In one embodiment, after step (c'), the method further comprises comparing the molecular weight value of raw PRP obtained in the purification process of Hib PRP with the molecular weight value of raw PRP calculated in step (c'). can do.
상기 특정 수준 이상의 원말 PRP의 분자량 분포를 만족하는, 상기 산출된 원말 PRP의 분자량은 다음과 같이 IPC (In Process Control)에 활용될 수 있다: PRP 원말에서 특정 수준 이상의 분자량 분포를 갖는 원말 PRP를 생산하기 위해서는, PRP 정제 최종 산물인 원말에서 원말 PRP의 분자량이, 상기 회귀 분석에 의해 산출된 원말 PRP 분자량 값을 가져야 한다. 따라서, PRP 정제 최종 산물에서 원말 PRP의 분자량이 상기 산출된 원말 PRP 분자량 값을 갖는지를 모니터링하여 PRP 생산의 IPC(In Process Control)에 활용할 수 있다. The calculated molecular weight of the raw PRP, which satisfies the molecular weight distribution of the raw PRP above the specific level, can be utilized for IPC (In Process Control) as follows: Produce raw PRP having a molecular weight distribution above a specific level in the PRP raw material In order to do this, the molecular weight of the raw PRP in the raw PRP purification final product must have the raw PRP molecular weight value calculated by the regression analysis. Therefore, by monitoring whether the molecular weight of raw PRP in the final product of PRP purification has the calculated raw PRP molecular weight value, it can be utilized for IPC (In Process Control) of PRP production.
상술된 본 발명의 방법에 따라 산출된 조 PRP의 분자량 값 및 원말 PRP의 분자량 값은 각각 단독으로 또는 서로 조합하여 본 발명의 PRP의 품질 평가 방법에 사용될 수 있다. The molecular weight value of crude PRP and the molecular weight value of raw PRP calculated according to the method of the present invention described above may be used alone or in combination with each other in the method for evaluating the quality of PRP of the present invention.
본 발명의 PRP 품질 평가 방법에 따르면, Hib의 PRP 정제 공정 단계에서 회귀분석을 통해 조 PRP의 분자량 값 또는 원말 PRP의 분자량 값을 측정함으로써, 최종적으로 생산되는 Hib PRP 제품의 품질을 평가하고 미리 예측할 수 있다. 또한, 본 발명의 방법은 Hib PRP 원말의 정제 공정 중간 단계에서 IPC (In Process Control) 방법으로 적용할 수 있으며, 실제 공정에서 발생하는 품질 이슈 시점을 확인하는데 유용하게 활용될 수 있다. According to the PRP quality evaluation method of the present invention, by measuring the molecular weight value of crude PRP or the molecular weight value of raw PRP through regression analysis in the PRP purification process step of Hib, the quality of the Hib PRP product finally produced can be evaluated and predicted in advance. can In addition, the method of the present invention can be applied as an IPC (In Process Control) method in the intermediate stage of the purification process of Hib PRP raw powder, and can be usefully utilized to confirm the timing of quality issues occurring in the actual process.
도 1은 배치(batch) 1 내지 배치(batch) 6에서 측정한 PRP 분자량 측정 데이터와 조(crude) PRP 분자량 측정 데이터를 보여준다. 1 shows PRP molecular weight measurement data and crude PRP molecular weight measurement data measured in batches 1 to 6.
도 2는 조 PRP 분자량과 원말 PRP 분자량 분포간의 회귀 분석을 행하여 얻은 적합선 및 회귀 방정식을 보여준다. 또한, 원말 PRP 분자량과 원말 PRP 분자량 분포간의 회귀 분석을 행하여 얻은 적합선 및 회귀 방정식을 보여준다. 2 shows a fitted line and a regression equation obtained by performing a regression analysis between the crude PRP molecular weight and the raw PRP molecular weight distribution. Also, the fitted line and regression equation obtained by performing regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution are shown.
도 3은 본 발명의 Hib의 PRP 생산 공정에서 IPC (In Process Control)을 행하는 개념을 보여주는 모식도이다. 3 is a schematic diagram showing the concept of performing IPC (In Process Control) in the PRP production process of Hib of the present invention.
이하에서 본 발명을 실시예에 의해 상세히 설명한다. 단 하기의 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 하기 실시예에 의해 한정되지 아니한다. Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.
실시예Example
실험예 1: 조 PRP 분자량 및 원말 PRP 분자량 분포를 이용한 품질 평가 방법 Experimental Example 1: Quality evaluation method using crude PRP molecular weight and raw PRP molecular weight distribution
실험예 1-1: 조 PRP의 분자량 측정 Experimental Example 1-1: Measurement of molecular weight of crude PRP
헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 PRP를 정제하여 최종 정제된 PRP 원말을 제조하는 공정 동안에, 정제 공정 중간 산물로, 정제가 완료되지 않은 조 PRP를 포함하는 샘플을 채취하고, 채취된 샘플내의 조 PRP의 분자량을 측정하였다. PRP는 높은 극성을 갖는 물질이므로 역상 크로마토그래피(reverse phase chromatography)의 컬럼을 통과하지만, PRP 이외의 다른 불순물은 역상 크로마토그래피 컬럼에 결합되므로, 역상 크로마토그래피를 이용하면 샘플내에서 PRP만을 순수하게 분리할 수 있다. 조 PRP를 포함하는 채취한 샘플을 역상 크로마토그래피 컬럼에 투입하여, 컬럼에 결합하지 않고 통과되어 나온 조 PRP에 대해, MALS (Multi-Angle Light Scattering, MALS) 검출기를 사용하여 조 PRP의 분자량을 측정하였다. During the process of purifying PRP from the culture solution of Haemophilus influenzae type b (Hib) to produce the final purified PRP raw material, as an intermediate product of the purification process, a sample containing unrefined crude PRP is used. It was collected and the molecular weight of the crude PRP in the collected sample was measured. Since PRP is a substance with high polarity, it passes through a column of reverse phase chromatography, but impurities other than PRP are bound to the reverse phase chromatography column. can do. The collected sample containing crude PRP is put into a reversed-phase chromatography column, and the molecular weight of crude PRP is measured using a MALS (Multi-Angle Light Scattering, MALS) detector for the crude PRP that has passed through without binding to the column. did
실험예 1-2: 원말 PRP의 분자량 분포 측정 Experimental Example 1-2: Measurement of molecular weight distribution of raw PRP
헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 PRP를 정제하여 최종 정제된 PRP 원말에서, 원말 PRP의 분자량 분포를 측정하였다. 원말 PRP의 분자량 분포는 다음 과정을 통해 측정하였다. 블루덱스트란(Blue Dextran)과 염화나트륨(Sodium Chloride, NaCl)를 분자량 표준 물질로 사용하여 크기 배제 크로마토 그래피(Size Exclusion Chromatography, SEC)를 통과시켜, 표준 물질의 머무름 시간(RT, retension time)를 확인하였다. 컬럼으로부터 첫번째로 나오는 블루덱스트란의 RT를 V0로 하고, 두번째로 나오는 염화나트륨의 RT를 Vt로 하여, 분배계수(Kd, Partition or Distribution Constant)가 0.3을 만족하는 기준값 Vp를 계산하였다[Ex: Vp = 0.3 X (Vt - V0) + V0)]. 이어서, 원말 PRP를 크기 배제 크로마토그래피(SEC)를 통과시켜 입자 크기 별로 분획하여 Main Peak의 전체 면적중에서 Vp 값을 기준으로 하여 앞에 나온 면적의 상대 면적(%)을 구하여 이를 분자량 분포로 표시하였다. 즉, 원말 PRP의 분자량 분포는 다음식으로 구하였다: 분자량 분포(%) = Vp 앞 부분의 면적/전체 면적 X 100. PRP was purified from the culture medium of Haemophilus influenzae type b (Hib), and the molecular weight distribution of raw PRP was measured in the final purified PRP raw material. The molecular weight distribution of raw PRP was measured through the following procedure. Using Blue Dextran and sodium chloride (NaCl) as molecular weight standards, pass size exclusion chromatography (SEC) to check the retention time (RT, retention time) of the standard material did A reference value Vp that satisfies the partition coefficient (Kd, Partition or Distribution Constant) of 0.3 was calculated by using the RT of blue dextran coming out first from the column as V0 and the RT of sodium chloride coming out second as Vt [Ex: Vp = 0.3 X (Vt - V0) + V0)]. Then, the raw PRP was passed through size exclusion chromatography (SEC), fractionated by particle size, and the relative area (%) of the preceding area was obtained based on the Vp value among the total area of the main peak, and this was expressed as a molecular weight distribution. That is, the molecular weight distribution of raw PRP was obtained by the following formula: molecular weight distribution (%) = area before Vp/total area X 100.
실험예 1-3: 회귀분석 Experimental Example 1-3: Regression analysis
상술한 실험예 1-1 및 실험예 1-2에 의한 측정을, Hib PRP 정제 공정의 배치 2 내지 배치 5의 총 4개의 배치에 대해 수행하고, 측정된 데이터를 토대로 회귀분석을 수행하였다. 구체적으로, 실험예 1-1에서 측정한 조 PRP의 분자량 데이터와, 실험예 1-2에서 측정한 원말 PRP의 분자량 분포 데이터를 각각 변수로 정하고, 선형 회귀 분석에 의해 회귀 방정식을 산출하였다(표 1 및 도 2). 산출된 회귀 방정식은 "y = 0.0042x + 0.3377"이었다. 상기 회귀 방정식에서 x는 조 PRP의 분자량를 나타내고, y는 원말 PRP의 분자량 분포를 나타내며, 회귀 방정식의 신뢰도(설명력)을 나타내는 R제곱값(R2)은 0.7346이었다. R제곱값(R2)이 0.7346을 나타내어 높은 상관관계를 보임을 확인하였다. 상기 회귀 방정식을 통해 원말 PRP 분자량 분포 50% 이상인 원말 PRP를 생산하기 위해서는 조 PRP의 분자량은 38.5 kDa 이상을 만족해야 함을 알 수 있다. 상기와 같은 분석을 토대로, PRP의 정제 공정중에서 조 PRP의 분자량을 38.5 kDa 이상이 되도록 관리하면, 품질 관리(quality control)기준이 되는, 원말 PRP의 분자량 분포가 50% 이상인, 최종 정제된 PRP 원말을 얻을 수 있다. Measurements according to Experimental Example 1-1 and Experimental Example 1-2 described above were performed for a total of four batches of batches 2 to 5 of the Hib PRP purification process, and regression analysis was performed based on the measured data. Specifically, the molecular weight data of crude PRP measured in Experimental Example 1-1 and the molecular weight distribution data of crude PRP measured in Experimental Example 1-2 were set as variables, respectively, and a regression equation was calculated by linear regression analysis (Table 1 and Figure 2). The calculated regression equation was "y = 0.0042x + 0.3377". In the regression equation, x represents the molecular weight of the crude PRP, y represents the molecular weight distribution of the raw PRP, and the R square value (R 2 ) representing the reliability (explanatory power) of the regression equation was 0.7346. It was confirmed that the R-squared value (R 2 ) was 0.7346, indicating a high correlation. Through the regression equation, it can be seen that the molecular weight of crude PRP must be 38.5 kDa or more in order to produce raw PRP having a molecular weight distribution of 50% or more of raw PRP. Based on the above analysis, if the molecular weight of crude PRP is managed to be 38.5 kDa or more during the purification process of PRP, the molecular weight distribution of raw PRP, which is a quality control standard, is 50% or more, the final purified PRP raw powder can get
Batch 2 Batch 2 Batch 3 Batch 3 Batch 4 Batch 4 Batch 5 Batch 5 회귀분석 산출값Regression analysis output
조 PRP 분자량Crude PRP molecular weight 128.6128.6 56.956.9 53.353.3 38.938.9 38.5 38.5
원말PRP 분자량 분포Raw PRP molecular weight distribution 85%85% 66%66% 64%64% 37%37% 50% 50%
분자량 단위: kDa Molecular Weight Unit: kDa
실험예 2: 원말 PRP의 분자량 및 분자량 분포를 이용한 품질 평가 방법 Experimental Example 2: Quality evaluation method using molecular weight and molecular weight distribution of raw PRP
실험예 2-1: 원말 PRP 분자량의 측정 Experimental Example 2-1: Measurement of raw PRP molecular weight
헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 PRP를 정제하여 최종 정제된 PRP 원말을 제조하였다. 최종 정제된 PRP 원말에서 원말 PRP의 분자량을 측정하였다. 원말 PRP의 분자량 측정은 실험예 1-1의 조 PRP 분자량 측정과 동일한 방법으로 수행하였다. 원말 PRP를 포함하는 샘플을 역상 크로마토그래피 컬럼에 투입하여, 컬럼에 결합하지 않고 통과되어 나온 원말 PRP에 대해, MALS (Multi-Angle Light Scattering, MALS) 검출기를 사용하여 원말 PRP의 분자량을 측정하였다. PRP was purified from the culture medium of Haemophilus influenzae type b (Hib) to prepare the final purified PRP raw material. In the final purified PRP raw material, the molecular weight of the raw PRP was measured. The molecular weight measurement of raw PRP was performed in the same manner as the crude PRP molecular weight measurement of Experimental Example 1-1. A sample containing raw PRP was put into a reverse phase chromatography column, and the molecular weight of raw PRP was measured using a MALS (Multi-Angle Light Scattering, MALS) detector for raw PRP that passed through without binding to the column.
실험예 2-2. PRP 원말의 분자량 분포 측정 Experimental Example 2-2. Molecular weight distribution measurement of PRP raw material
헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 PRP를 정제하여 최종 정제된 PRP 원말에서, 원말 PRP의 분자량 분포를 측정하였다. 원말 PRP의 분자량 분포는 상기 실험예 1-2에서 설명된 방법과 동일한 방법으로 측정하였다. PRP was purified from the culture medium of Haemophilus influenzae type b (Hib), and the molecular weight distribution of the raw PRP was measured in the final purified PRP raw material. The molecular weight distribution of raw PRP was measured in the same manner as described in Experimental Example 1-2.
실험예 2-3. 회귀분석 Experimental Example 2-3. regression analysis
상술한 실험예 2-1 및 실험예 2-2에 의한 측정을 Hib PRP 정제 공정의 배치 1 내지 배치 6의 총 6개의 배치에 대해 수행하고, 측정된 데이터를 토대로 회귀분석을 수행하였다. 구체적으로, 실험예 2-1에서 측정한 원말 PRP의 분자량 데이터와, 실험예 2-2에서 측정한 원말 PRP의 분자량 분포 데이터를 각각 변수로 정하고, 선형 회귀 분석에 의해 회귀 방정식을 산출하였다(표 2 및 도 2). 산출된 회귀 방정식은 "y = 0.0131x - 0.4035"이었다. 상기 회귀 방정식에서 x는 원말 PRP의 분자량을 나타내고, y는 원말 PRP의 분자량 분포를 나타내며, 회귀 방정식의 신뢰도(설명력)을 나타내는 R제곱값(R2)은 0.9222이었다. R제곱값(R2)이 0.9222을 나타내어 높은 상관관계를 보임을 확인하였다. 상기 회귀 방정식을 통해 원말 PRP의 분자량 분포 50% 이상인 원말 PRP를 생산하기 위해서는 원말 PRP의 분자량은 69.1 kDa 이상을 만족해야 함을 알 수 있다. 상기와 같은 분석을 토대로, PRP의 정제 공정중에서 원말 PRP의 분자량을 69.1 kDa 이상이 되도록 관리하면, 품질관리(quality control)기준이 되는, 원말 PRP의 분자량 분포가 50% 이상인, 최종 정제된 PRP 원말을 얻을 수 있다. Measurements according to Experimental Example 2-1 and Experimental Example 2-2 described above were performed for a total of 6 batches of batches 1 to 6 of the Hib PRP purification process, and regression analysis was performed based on the measured data. Specifically, the molecular weight data of the raw PRP measured in Experimental Example 2-1 and the molecular weight distribution data of the raw PRP measured in Experimental Example 2-2 were set as variables, respectively, and a regression equation was calculated by linear regression analysis (Table 2 and Fig. 2). The calculated regression equation was "y = 0.0131x - 0.4035". In the regression equation, x represents the molecular weight of the raw PRP, y represents the molecular weight distribution of the raw PRP, and the R square value (R 2 ) representing the reliability (explanatory power) of the regression equation was 0.9222. It was confirmed that the R-squared value (R 2 ) was 0.9222, indicating a high correlation. It can be seen from the regression equation that the molecular weight of raw PRP must be 69.1 kDa or more in order to produce raw PRP having a molecular weight distribution of 50% or more of raw PRP. Based on the above analysis, if the molecular weight of raw PRP is managed to be 69.1 kDa or more during the purification process of PRP, the molecular weight distribution of raw PRP, which is a quality control standard, is 50% or more, the final purified PRP raw material can get
Batch 1 Batch 1 Batch 2 Batch 2 Batch 3 Batch 3 Batch 4 Batch 4 Batch 5 Batch 5 Batch 6 Batch 6 회귀분석
산출값regression analysis
output
원말PRP분자량Raw PRP molecular weight 72.172.1 99.899.8 76.576.5 75.575.5 64.564.5 5555 69.1 69.1
원말PRP분자량
분포Raw PRP molecular weight
Distribution 		57%57% 85%85% 66%66% 64%64% 37%37% 29%29% 50% 50%
분자량 단위: kDa Molecular Weight Unit: kDa
상기에서는 본 출원의 대표적인 실시예를 예시적으로 설명하였으나, 본 출원의 범위는 상기와 같은 특정 실시예만 한정되지 아니하며, 해당 분야에서 통상의 지식을 가진 자라면 본 출원의 청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다. In the above, representative embodiments of the present application have been exemplarily described, but the scope of the present application is not limited only to the specific embodiments as described above, and those of ordinary skill in the art are within the scope described in the claims of the present application. can be changed appropriately.

Claims (12)

  1. 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법: A method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the steps of:
    (a) 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하는 공정 중에서 조(crude) PRP를 포함하는 샘플을 채취하는 단계; (a) In the process of purifying polyribosyl ribitol phosphate (PRP) from a culture solution of Haemophilus influenzae type b (Hib), a sample containing crude PRP is collected step;
    (b) 채취한 샘플 내의 조 PRP의 분자량을 측정하는 단계; (b) measuring the molecular weight of the crude PRP in the collected sample;
    (c) PRP를 최종 정제한 원말에서 원말 PRP의 분자량 분포를 측정하는 단계; (c) measuring the molecular weight distribution of the raw PRP in the final purified raw PRP;
    (d) 단계 (b)에서 측정한 조 PRP의 분자량 데이터와, 단계 (c)에서 측정한 원말 PRP의 분자량 분포 데이터를 사용하여, 조 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (d) using the molecular weight data of the crude PRP measured in step (b) and the molecular weight distribution data of the crude PRP measured in step (c), a regression analysis between the molecular weight of the crude PRP and the molecular weight distribution of the crude PRP was performed performing; and
    (e) 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 조 PRP 분자량을 산출하는 단계. (e) calculating a crude PRP molecular weight that satisfies the original PRP molecular weight distribution above a certain level from the regression analysis.
  2. 청구항 1에 있어서, The method according to claim 1,
    상기 단계 (b)에서 조 PRP의 분자량의 측정은 이온교환 크로마토그래피, 친화성 크로마토그래피, 크기배제 크로마토그래피, 역상 크로마토그래피, 또는 소수성 상호작용 크로마토그래피를 통해 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법.The measurement of the molecular weight of the crude PRP in step (b) is carried out through ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography, purification process of PRP of Hib Methods for evaluating the quality of PRP in
  3. 청구항 2에 있어서, 3. The method according to claim 2,
    상기 단계 (b)에서 조 PRP의 분자량의 측정은 자외선 흡광도 검출 방법 또는 광산란 검출 방법을 더 포함하여 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. The measurement of the molecular weight of the crude PRP in the step (b) is a method for evaluating the quality of PRP in the purification process of PRP of Hib, which is performed by further including an ultraviolet absorbance detection method or a light scattering detection method.
  4. 청구항 1에 있어서, The method according to claim 1,
    상기 단계 (c)에서 원말 PRP의 분자량 분포의 측정은 크로마토그래피 분석 방법을 이용하여 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. In the step (c), the measurement of the molecular weight distribution of the raw PRP is performed using a chromatography analysis method, a method for evaluating the quality of PRP in the PRP purification process of Hib.
  5. 청구항 1에 있어서, The method according to claim 1,
    상기 단계 (d)에서 회귀 분석은 선형 회귀 분석인 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. In the step (d), the regression analysis is a linear regression analysis, a method for evaluating the quality of PRP in the purification process of PRP of Hib.
  6. 청구항 1에 있어서, The method according to claim 1,
    상기 단계 (e) 이후에, Hib의 PRP의 정제 공정에서 얻어지는 조 PRP의 분자량 값과, 상기 단계 (e)에서 산출된 조 PRP의 분자량 값을 비교하는 단계를 더 포함하는, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. Purification of PRP of Hib, further comprising, after step (e), comparing the molecular weight value of crude PRP obtained in the purification process of PRP of Hib with the molecular weight value of crude PRP calculated in step (e) Methods for evaluating the quality of PRP in the process.
  7. 다음의 단계를 포함하는 Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법: A method for evaluating the quality of PRP in the purification process of PRP of Hib, comprising the steps of:
    (a') 헤모필루스 인플루엔자 타입 b (Haemophilus influenzae type b, Hib)를 배양한 배양액으로부터 폴리리보실 리비톨 포스페이트 (polyribosyl ribitol phosphate, PRP)를 정제하여 얻은 최종 정제 원말에서, 원말 PRP의 분자량 및 원말 PRP의 분자량 분포를 측정하는 단계; (a') In the final purified raw material obtained by purifying polyribosyl ribitol phosphate (PRP) from the culture medium of Haemophilus influenzae type b (Hib), the molecular weight of the raw PRP and the raw PRP measuring the molecular weight distribution of
    (b') 단계 (a')에서 측정한 원말 PRP의 분자량 데이터와 원말 PRP의 분자량 분포 데이터를 사용하여, 원말 PRP 분자량과 원말 PRP 분자량 분포 간의 회귀 분석(regression analysis)을 수행하는 단계; 및 (b') using the molecular weight data of the raw PRP and the molecular weight distribution data of the raw PRP measured in step (a'), performing a regression analysis between the raw PRP molecular weight and the raw PRP molecular weight distribution; and
    (c') 회귀 분석으로부터 특정 수준 이상의 원말 PRP 분자량 분포를 만족하는 원말 PRP 분자량을 산출하는 단계. (c') calculating the raw PRP molecular weight satisfying the raw PRP molecular weight distribution of a specific level or higher from the regression analysis.
  8. 청구항 7에 있어서, 8. The method of claim 7,
    상기 단계 (a')에서 원말 PRP의 분자량 측정은 이온교환 크로마토그래피, 친화성 크로마토그래피, 크기배제 크로마토그래피, 역상 크로마토그래피, 또는 소수성 상호작용 크로마토그래피 방법을 통해 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. In the step (a'), the molecular weight of the raw PRP is measured through ion exchange chromatography, affinity chromatography, size exclusion chromatography, reverse phase chromatography, or hydrophobic interaction chromatography method. Purification of PRP of Hib Methods for evaluating the quality of PRP in the process.
  9. 청구항 8에 있어서, 9. The method of claim 8,
    상기 단계 (a')에서 원말 PRP의 분자량 측정은 자외선 흡광도 검출 방법 또는 광산란 검출 방법을 더 포함하여 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. In the step (a'), the molecular weight measurement of the raw PRP is performed by further including an ultraviolet absorbance detection method or a light scattering detection method, a method for evaluating the quality of PRP in the PRP purification process of Hib.
  10. 청구항 7에 있어서, 8. The method of claim 7,
    상기 단계 (a')에서 원말 PRP의 분자량 분포의 측정은 크로마토그래피 분석 방법을 이용하여 행하는 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. In the step (a'), the measurement of the molecular weight distribution of the raw PRP is performed using a chromatography analysis method, a method for evaluating the quality of PRP in the PRP purification process of Hib.
  11. 청구항 7에 있어서, 8. The method of claim 7,
    상기 단계 (b')에서 회귀 분석은 선형 회귀 분석인 것인, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. The regression analysis in the step (b') is a linear regression analysis, a method for evaluating the quality of PRP in the purification process of PRP of Hib.
  12. 청구항 7에 있어서, 8. The method of claim 7,
    상기 단계 (c') 이후에, Hib의 PRP의 정제 공정에서 얻어지는 원말 PRP의 분자량 값과, 상기 단계 (c')에서 산출된 원말 PRP의 분자량 값을 비교하는 단계를 더 포함하는, Hib의 PRP의 정제 공정에서의 PRP의 품질 평가 방법. After the step (c'), the PRP of Hib further comprising the step of comparing the molecular weight value of the raw PRP obtained in the purification process of the PRP of Hib with the molecular weight value of the raw PRP calculated in the step (c') A method for evaluating the quality of PRP in the purification process of
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