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Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines. CHO细胞聚类法监测无细胞百日咳疫苗中百日咳毒素活性的可转移性研究。
Q4 Medicine Pub Date : 2016-01-01
R Isbrucker, A Daas, L Wagner, A Costanzo

Current regulations for acellular pertussis (aP) vaccines require that they are tested for the presence of residual or reversion-derived pertussis toxin (PTx) activity using the mouse histamine sensitisation test (HIST). Although a CHO cell clustering assay can be used by manufacturers to verify if sufficient inactivation of the substance has occurred in-process, this assay cannot be used at present for the final product due to the presence of aluminium adjuvants which interfere with mammalian cell cultures. Recently, 2 modified CHO cell clustering assays which accommodate for the adjuvant effects have been proposed as alternatives to the HIST. These modified assays eliminate the adjuvant-induced cytotoxicity either through dilution of the vaccine (called the Direct Method) or by introducing a porous barrier between the adjuvant and the cells (the Indirect Method). Transferability and suitability of these methods for testing of products present on the European market were investigated during a collaborative study organised by the European Directorate for the Quality of Medicines & HealthCare (EDQM). Thirteen laboratories participated in this study which included 4 aP-containing vaccines spiked by addition of PTx. This study also assessed the transferability of a standardised CHO cell clustering assay protocol for use with non-adjuvanted PTx preparations. Results showed that the majority of laboratories were able to detect the PTx spike in all 4 vaccines at concentrations of 4 IU/mL or lower using the Indirect Method. This sensitivity is in the range of the theoretical sensitivity of the HIST. The Direct Method however did not show the expected results and would need additional development work.

目前关于无细胞百日咳(aP)疫苗的法规要求使用小鼠组胺致敏试验(HIST)检测它们是否存在残留或逆转性百日咳毒素(PTx)活性。尽管制造商可以使用CHO细胞聚类分析来验证过程中是否发生了物质的充分失活,但由于铝佐剂的存在会干扰哺乳动物细胞培养,因此目前该分析不能用于最终产品。最近,提出了两种适应佐剂作用的改进的CHO细胞聚类试验作为HIST的替代方法。通过稀释疫苗(称为直接法)或在佐剂和细胞之间引入多孔屏障(间接法),这些改良的检测方法消除了佐剂诱导的细胞毒性。欧洲药品和保健质量理事会(EDQM)组织了一项合作研究,调查了这些方法在欧洲市场上检测产品的可转移性和适用性。13个实验室参与了这项研究,其中包括4种添加了PTx的含ap疫苗。本研究还评估了用于非佐剂PTx制剂的标准化CHO细胞聚类分析方案的可转移性。结果表明,大多数实验室能够在4 IU/mL或更低浓度的4种疫苗中检测到PTx尖峰。该灵敏度在HIST的理论灵敏度范围内。然而,直接方法没有显示预期的结果,需要额外的开发工作。
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引用次数: 0
Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement. 无细胞百日咳疫苗的 HIST 替代品:替代品的进展与挑战。
Q4 Medicine Pub Date : 2016-01-01
J Arciniega, L Wagner, R Prymula, P Sebo, R Isbrucker, B Descampe, J M Chapsal, A Costanzo, C Hendriksen, M Hoonaker, S Nelson, K Lidster, W Casey, D Allen

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

细胞百日咳疫苗小鼠组胺致敏试验替代品国际研讨会:作为第九届世界生命科学替代品和动物使用大会的卫星会议,"细胞百日咳疫苗小鼠组胺致敏试验替代品:HIST替代品的进展与挑战 "国际研讨会于2014年8月24日在捷克共和国布拉格举行。与会者讨论了作为无细胞百日咳疫苗组胺致敏试验(HIST)替代品的体外检测方法的开发、验证和实施方面的进展和挑战。讨论的重点是一致性方法、监管机构接受统一方法的必要框架,以及最近国际上为开发体外检测方法以取代组胺致敏试验(HIST)所做的努力。研讨会与会者一致认为,可接受的 HIST 替代品应以 ADP 核糖介导的细胞中毒为基础,因此应进一步研究和开发可测量细胞中毒的 CHO 细胞聚类检测法,作为 HIST 的可能替代品。与会者还同意继续进行有国家和国际标准化机构参与的多国讨论,以达成共识,并在此背景下组织合作研究,以确定化验特性和校准参考材料。
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引用次数: 0
Collaborative study for the validation of an improved HPLC assay for recombinant IFN-alfa-2. 重组ifn - α -2的高效液相色谱检测方法的验证。
Q4 Medicine Pub Date : 2016-01-01
K H Jönsson, A Daas, K H Buchheit, E Terao

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

目前的欧洲药典(Ph. Eur.)关于干扰素(IFN)- α -2的文本包括使用白蛋白作为校准器的非特异性光度蛋白测定和用于确定保护作用效价的高度可变的基于细胞的测定。官方药物控制实验室(OMCLs)要求改进重组干扰素α -2原料药的批量控制方法和成品的市场监测检测方法,包括用人血清白蛋白(HSA)配制的产品。瑞典医疗产品管理局(MPA)开发了一种HPLC法,用于检测ifn - α -2产品。生物标准化计划(BSP)的初步合作研究;研究代码BSP039)显示需要进行微小的更改以提高校准曲线的线性,分析重现性和稳健性。该合作研究的代码为BSP071,目的是转移并进一步验证这种改进的HPLC方法。十个实验室参与了这项研究。四种已上市的ifn - α -2制剂(一种含有HSA)连同Ph. Eur。采用ifn - α -2a和ifn - α -2b的化学标准物质(CRS)和两家生产厂家的内部标准品进行定量分析。改进的方法被成功地转移到所有实验室,尽管设备的局部变化。与BSP039研究的结果相比,ifn - α -2的主要形式和氧化形式之间的分辨率得到了提高。改进的方法甚至允许在主峰之后的额外峰的部分分辨率。在所有实验室的所有样品中,IFN主峰的对称性均可接受。用Ph. Eur.建立校准曲线。IFN-alfa-2a和IFN-alfa-2b具有良好的线性关系,截距接近原点,决定系数大于0.9995。测定的重复性、中间精密度和重现性随被测样品在可接受范围内的变化而变化。尽管没有共同的参考制剂,但通过比较参与者获得的值与制造商确定的声明内容来估计的测试准确性是良好的。总之,本研究表明,新方法适用、重现性好、可转移性好。修订欧洲博士学位的建议。文本呈现。
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引用次数: 0
Alternative to Ph. Eur. pour-plate method for detection of microbial contamination in non-sterile pharmaceutical preparations. Ph. Eur的替代品。非无菌药物制剂中微生物污染检测的倾板法。
Q4 Medicine Pub Date : 2016-01-01
A Palicz, A Paul, A Hofmann, K Denzel

The current European Pharmacopoeia (Ph. Eur.) texts for Interferon (IFN)-alfa-2 include a nonspecific photometric protein assay using albumin as calibrator and a highly variable cell-based assay for the potency determination of the protective effects. A request was expressed by the Official Medicines Control Laboratories (OMCLs) for improved methods for the batch control of recombinant interferon alfa-2 bulk and market surveillance testing of finished products, including those formulated with Human Serum Albumin (HSA). A HPLC method was developed at the Medical Products Agency (MPA, Sweden) for the testing of IFN-alfa-2 products. An initial collaborative study run under the Biological Standardisation Programme (BSP; study code BSP039) revealed the need for minor changes to improve linearity of the calibration curves, assay reproducibility and robustness. The goal of the collaborative study, coded BSP071, was to transfer and further validate this improved HPLC method. Ten laboratories participated in the study. Four marketed IFN-alfa-2 preparations (one containing HSA) together with the Ph. Eur. Chemical Reference Substance (CRS) for IFN-alfa-2a and IFN-alfa-2b, and in-house reference standards from two manufacturers were used for the quantitative assay. The modified method was successfully transferred to all laboratories despite local variation in equipment. The resolution between the main and the oxidised forms of IFN-alfa-2 was improved compared to the results from the BSP039 study. The improved method even allowed partial resolution of an extra peak after the principal peak. Symmetry of the main IFN peak was acceptable for all samples in all laboratories. Calibration curves established with the Ph. Eur. IFN-alfa-2a and IFN-alfa-2b CRSs showed excellent linearity with intercepts close to the origin and coefficients of determination greater than 0.9995. Assay repeatability, intermediate precision and reproducibility varied with the tested sample within acceptable ranges. Test accuracy estimated by comparing the values obtained by the participants to the declared contents determined by the manufacturers was good despite the absence of a common reference preparation. In conclusion, the present study showed that the new method is suitable, reproducible and transferable. Proposals for the revision of Ph. Eur. texts are presented.

目前的欧洲药典(Ph. Eur.)关于干扰素(IFN)- α -2的文本包括使用白蛋白作为校准器的非特异性光度蛋白测定和用于确定保护作用效价的高度可变的基于细胞的测定。官方药物控制实验室(OMCLs)要求改进重组干扰素α -2原料药的批量控制方法和成品的市场监测检测方法,包括用人血清白蛋白(HSA)配制的产品。瑞典医疗产品管理局(MPA)开发了一种HPLC法,用于检测ifn - α -2产品。生物标准化计划(BSP)的初步合作研究;研究代码BSP039)显示需要进行微小的更改以提高校准曲线的线性,分析重现性和稳健性。该合作研究的代码为BSP071,目的是转移并进一步验证这种改进的HPLC方法。十个实验室参与了这项研究。四种已上市的ifn - α -2制剂(一种含有HSA)连同Ph. Eur。采用ifn - α -2a和ifn - α -2b的化学标准物质(CRS)和两家生产厂家的内部标准品进行定量分析。改进的方法被成功地转移到所有实验室,尽管设备的局部变化。与BSP039研究的结果相比,ifn - α -2的主要形式和氧化形式之间的分辨率得到了提高。改进的方法甚至允许在主峰之后的额外峰的部分分辨率。在所有实验室的所有样品中,IFN主峰的对称性均可接受。用Ph. Eur.建立校准曲线。IFN-alfa-2a和IFN-alfa-2b具有良好的线性关系,截距接近原点,决定系数大于0.9995。测定的重复性、中间精密度和重现性随被测样品在可接受范围内的变化而变化。尽管没有共同的参考制剂,但通过比较参与者获得的值与制造商确定的声明内容来估计的测试准确性是良好的。总之,本研究表明,新方法适用、重现性好、可转移性好。修订欧洲博士学位的建议。文本呈现。
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引用次数: 0
Collaborative study for the calibration of replacement batches for the heparin low-molecular-mass for assay biological reference preparation. 用于测定生物参比制剂的肝素低分子质量替代批的校准合作研究。
Q4 Medicine Pub Date : 2016-01-01
E Terao, A Daas

The European Pharmacopoeia (Ph. Eur.) prescribes the control of the activity of low molecular mass heparins by assays for anti-Xa and anti-IIa activities (monograph 0828), using a reference standard calibrated in International Units (IU). An international collaborative study coded BSP133 was launched in the framework of the Biological Standardisation Programme (BSP) run under the aegis of the Council of Europe and the European Commission to calibrate replacement batches for the dwindling stocks of the Heparin low-molecular-mass for assay Biological Reference Preparation (BRP) batch 8. Thirteen official medicines control and manufacturers laboratories from European and non-European countries took part in this study to calibrate two freeze-dried candidate batches against the 3rd International Standard (IS) for heparin, low molecular weight (11/176; 3rd IS). The Heparin low-molecular-mass for assay BRP (batch 8) was also included in the test panel to check the continuity between subsequent BRP batches. Taking into account the stability data, the results of this collaborative study and on the basis of the central statistical analysis performed at the European Directorate for the Quality of Medicines & HealthCare (EDQM), the 2 candidate batches were officially adopted by the Commission of the European Pharmacopoeia as Heparin low-molecular-mass for assay BRP batches 9 and 10 with assigned anti-Xa activities of 102 and 100 IU/vial and anti-IIa activities of 34 and 33 IU/vial respectively.

欧洲药典(Ph. Eur.)规定了通过测定抗xa和抗iia活性来控制低分子质量肝素的活性(专著0828),使用以国际单位(IU)校准的参考标准。编号为BSP133的国际合作研究在欧洲理事会和欧盟委员会主持下的生物标准化计划(BSP)框架内启动,以校准用于测定的肝素低分子质量生物参比制剂(BRP)第8批库存减少的替代批次。来自欧洲和非欧洲国家的13个官方药物控制和制造商实验室参与了这项研究,根据肝素第三国际标准(IS)校准两个冻干候选批次,低分子量(11/176;3)。用于BRP测定的肝素低分子质量(第8批)也包括在测试面板中,以检查后续BRP批次之间的连续性。考虑到稳定性数据、本合作研究的结果以及在欧洲药品和保健质量理事会(EDQM)进行的中心统计分析的基础上,2个候选批次被欧洲药典委员会正式采用为肝素低分子质量,用于BRP第9批和第10批,指定抗xa活性分别为102和100 IU/瓶,抗iia活性分别为34和33 IU/瓶。
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引用次数: 0
European Pharmacopoeia biological reference preparation for poliomyelitis vaccine (inactivated): collaborative study for the establishment of batch No. 3. 欧洲药典脊髓灰质炎灭活疫苗生物参比制剂:建立第3批的合作研究。
Q4 Medicine Pub Date : 2016-01-01
J Martin, A Daas, C Milne

Inactivated poliomyelitis vaccines are an important part of the World Health Organization (WHO) control strategy to eradicate poliomyelitis. Requirements for the quality control of poliomyelitis vaccines (inactivated) include the use of an in vitro D antigen quantification assay for potency determination on the final lot as outlined in the European Pharmacopoeia (Ph. Eur.) monograph 0214. Performance of this assay requires a reference preparation calibrated in International Units (IU). A Ph. Eur. biological reference preparation (BRP) for poliomyelitis vaccine (inactivated) calibrated in IU has been established for this purpose. Due to the dwindling stocks of batch 2 of the BRP a collaborative study was run as part of the European Directorate for the Quality of Medicines & HealthCare (EDQM) Biological Standardisation Programme to establish BRP batch 3 (BRP3). Twelve laboratories including Official Medicines Control Laboratories (OMCLs) and manufacturers participated. The candidate BRP3 (cBRP3) was from the same source and had the same characteristics as BRP batch 2 (BRP2). During the study the candidate was calibrated against the 3rd International Standard for inactivated poliomyelitis vaccine using in-house D antigen ELISA assays in line with the Ph. Eur. monograph 0214. The candidate was also compared to BRP2 to evaluate the continuity. Based on the results of the study, values of 320 DU/mL, 78 DU/mL and 288 DU/mL (D antigen units/mL) (IU) for poliovirus type 1, 2 and 3 respectively were assigned to the candidate. In June 2016, the Ph. Eur. Commission adopted the material as Ph. Eur. BRP for poliomyelitis vaccine (inactivated) batch 3.

灭活脊髓灰质炎疫苗是世界卫生组织根除脊髓灰质炎控制战略的重要组成部分。脊髓灰质炎疫苗(灭活疫苗)的质量控制要求包括使用体外D抗原定量测定法对最终批次进行效价测定,如欧洲药典(Ph. Eur.)专著0214所述。该分析的性能需要以国际单位(IU)校准的参比制剂。博士学位。为此目的建立了以国际单位校准的脊髓灰质炎灭活疫苗生物参比制剂(BRP)。由于第2批BRP的库存减少,作为欧洲药品和保健质量理事会(EDQM)生物标准化计划的一部分,开展了一项合作研究,以建立第3批BRP (BRP3)。包括官方药物控制实验室(omcl)和制造商在内的12个实验室参与了研究。候选BRP3 (cBRP3)与第2批BRP (BRP2)来源相同,具有相同的特征。在研究期间,使用符合Ph. Eur的内部D抗原ELISA测定,根据第三个灭活脊髓灰质炎疫苗国际标准对候选物进行校准。0214年专著。还将候选基因与BRP2进行比较,以评估其连续性。根据研究结果,该候选人的1型、2型和3型脊髓灰质炎病毒的D抗原单位(IU)分别为320 DU/mL、78 DU/mL和288 DU/mL。2016年6月,欧洲博士学位。委员会通过该材料为Ph. Eur。脊髓灰质炎BRP(灭活疫苗)第3批。
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引用次数: 0
Elaborating European Pharmacopoeia monographs for biotherapeutic proteins using substances from a single source. 使用单一来源的物质为生物治疗蛋白编写欧洲药典专著。
Q4 Medicine Pub Date : 2016-01-01
M Buda, S Wicks, E Charton

For more than twenty years, the European Pharmacopoeia (Ph. Eur.) monographs for biotherapeutic proteins have been elaborated using the multisource approach (Procedure 1), which has led to robust quality standards for many of the first-generation biotherapeutics. In 2008, the Ph. Eur. opened up the way towards an alternative mechanism for the elaboration of monographs (Procedure 4-BIO pilot phase), which is applied to substances still under patent protection, based on a close collaboration with the Innovator company, to ensure a harmonised global standard and strengthen the quality of the upcoming products. This article describes the lessons learned during the P4-BIO pilot phase and addresses the current thinking on monograph elaboration in the field of biotherapeutics. Case studies are described to illustrate the standardisation challenges associated with the complexity of biotherapeutics and of analytical procedures, as well as the approaches that help ensure expectations are met when setting monograph specifications and allow for compatibility with the development of biosimilars. Emphasis is put on monograph flexibility, notably by including tests that measure process-dependent microheterogeneity (e.g. glycosylation) in the Production section of the monograph. The European Pharmacopoeia successfully concluded the pilot phase of the P4-BIO during its 156th session on 22-23 November 2016.

二十多年来,欧洲药典(Ph. Eur.)生物治疗蛋白专著已经使用多源方法(程序1)进行了详细阐述,这导致了许多第一代生物治疗药物的强大质量标准。2008年,欧元博士学位。在与Innovator公司密切合作的基础上,开辟了专论制定的替代机制(程序4-BIO试点阶段),该机制适用于仍受专利保护的物质,以确保统一的全球标准并加强即将推出的产品的质量。本文描述了在P4-BIO试点阶段学到的经验教训,并解决了目前在生物治疗领域对专著阐述的思考。案例研究描述了与生物治疗和分析程序的复杂性相关的标准化挑战,以及帮助确保在设定专论规范时满足期望并允许与生物仿制药开发兼容的方法。重点放在各论的灵活性上,特别是通过在各论的生产部分包括测量过程依赖的微观异质性(例如糖基化)的测试。欧洲药典在2016年11月22日至23日举行的第156届会议上成功结束了P4-BIO的试点阶段。
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引用次数: 0
In search of acceptable alternatives to the murine histamine sensitisation test (HIST): what is possible and practical? 寻找可接受的替代小鼠组胺致敏试验(HIST):什么是可能的和实用的?
Q4 Medicine Pub Date : 2016-01-01
L Wagner, R Isbrucker, C Locht, J Arciniega, A Costanzo, R McFarland, H Oh, M Hoonakker, J Descamps, S R Andersen, R K Gupta, K Markey, J M Chapsal, K Lidster, W Casey, D Allen

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: In Search of Acceptable Alternatives to the Murine Histamine Sensitization Test (HIST): What is Possible and Practical?' was held on 4 and 5 March 2015 in London, United Kingdom. Participants discussed the results of the data generated from an international collaborative study (BSP114 Phase 2) sponsored by the European Directorate for the Quality of Medicines & Health Care (EDQM) to determine if a modified Chinese hamster ovary (CHO) cell-based clustering assay is a suitable alternative to replace HIST. Workshop participants agreed that protocol transferability demonstrated in the collaborative study indicates that a standardised CHO cell assay is adequate for measuring pure PTx in reference preparations. However, vaccine manufacturers would still need to demonstrate that the method is valid to detect or measure residual PTx in their specific adjuvanted products. The 2 modified CHO cell protocols included in the study (the Direct and the Indirect Methods) deserve further consideration as alternatives to HIST. Using the CHO cell assay, an in vitro alternative, for acellular pertussis (aP) vaccine batch release testing would reduce the number of animals used for aP vaccine safety testing. A strategic, stepwise adoption plan was proposed, in which the alternative test would be used for release purposes first, and then, once sufficient confidence in its suitable performance has been gained, its use would be extended to stability testing.

无细胞百日咳疫苗小鼠组胺致敏试验替代品国际研讨会:寻找小鼠组胺致敏试验(HIST)的可接受替代品:什么是可能的和实用的?,于2015年3月4日至5日在英国伦敦举行。与会者讨论了由欧洲药品和卫生保健质量理事会(EDQM)赞助的一项国际合作研究(BSP114二期)产生的数据结果,以确定改良的中国仓鼠卵巢(CHO)细胞聚类分析是否适合替代HIST。研讨会参与者一致认为,合作研究中证明的方案可转移性表明,标准化的CHO细胞测定法足以测量参比制剂中的纯PTx。然而,疫苗制造商仍然需要证明该方法对检测或测量其特定佐剂产品中的残留PTx是有效的。研究中包括的两种修改后的CHO细胞方案(直接法和间接法)值得进一步考虑作为HIST的替代方案。使用CHO细胞测定法(一种体外替代方法)进行无细胞百日咳(aP)疫苗批量释放试验将减少用于aP疫苗安全性试验的动物数量。提出了一个战略性的、逐步采用的计划,其中替代测试将首先用于发布目的,然后,一旦对其合适的性能获得了足够的信心,它的使用将扩展到稳定性测试。
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引用次数: 0
Interpretation of size-exclusion chromatography for the determination of molecular-size distribution of human immunoglobulins. 测定人免疫球蛋白分子大小分布的粒径排除色谱解释。
Q4 Medicine Pub Date : 2016-01-01
S Christians, S Schluender, N D van Treel, M-E Behr-Gross

Molecular-size distribution by size-exclusion chromatography (SEC) [1] is used for the quantification of unwanted aggregated forms in therapeutic polyclonal antibodies, referred to as human immunoglobulins (Ig) in the European Pharmacopoeia. Considering not only the requirements of the monographs for human normal Ig (0338, 0918 and 2788) [2-4], but also the general chapter on chromatographic techniques (2.2.46) [5], several chromatographic column types are allowed for performing this test. Although the EDQM knowledge database gives only 2 examples of suitable columns as a guide for the user, these monographs permit the use of columns with different lengths and diameters, and do not prescribe either particle size or pore size, which are considered key characteristics of SEC columns. Therefore, the columns used may differ significantly from each other with regard to peak resolution, potentially resulting in ambiguous peak identity assignment. In some cases, this may even lead to situations where the manufacturer and the Official Medicines Control Laboratory (OMCL) in charge of Official Control Authority Batch Release (OCABR) have differing molecular-size distribution profiles for aggregates of the same batch of Ig, even though both laboratories follow the requirements of the relevant monograph. In the present study, several formally acceptable columns and the peak integration results obtained therewith were compared. A standard size-exclusion column with a length of 60 cm and a particle size of 10 µm typically detects only 3 Ig fractions, namely monomers, dimers and polymers. This column type was among the first reliable HPLC columns on the market for this test and very rapidly became the standard for many pharmaceutical manufacturers and OMCLs for batch release testing. Consequently, the distribution of monomers, dimers and polymers was established as the basis for the interpretation of the results of the molecular-size distribution test in the relevant monographs. However, modern columns with a smaller particle size provide better resolution and also reveal a class of components designated here as oligomers. This publication addresses the interpretation of the SEC test for Ig with respect to the following questions: - how can molecular-size distribution tests benefit from the use of the most recent column technology without changing the sense of well-established quality parameters? - is it possible to mathematically define a way to interpret chromatograms generated with various column types with the same fractionation range but different resolution power? - how should oligomers be considered regarding compliance with compendial specifications?

通过大小排除色谱(SEC)的分子大小分布[1]用于定量治疗性多克隆抗体中不需要的聚集形式,在欧洲药典中称为人免疫球蛋白(Ig)。考虑到人正常Ig各论(0338,0918和2788)[2-4]的要求,以及色谱技术通章(2.2.46)[5]的要求,本试验允许使用几种色谱柱类型。虽然EDQM知识数据库只给出了2个合适的柱作为用户指南的例子,这些专著允许使用不同长度和直径的柱,并且不规定颗粒大小或孔径,这被认为是SEC柱的关键特征。因此,所使用的列在峰分辨率方面可能会有很大的不同,这可能会导致峰标识分配不明确。在某些情况下,这甚至可能导致制造商和负责官方药品控制机构批次放行(OCABR)的官方药物控制实验室(OMCL)对同一批次Ig的聚集体具有不同的分子大小分布曲线,即使两个实验室都遵循相关专论的要求。在本研究中,比较了几种形式可接受的列及其得到的峰积分结果。标准尺寸排除柱长度为60 cm,粒径为10 μ m,通常仅检测3个Ig组分,即单体,二聚体和聚合物。该色谱柱类型是市场上首批可靠的高效液相色谱柱之一,并迅速成为许多制药企业和omcl的批释放测试标准。因此,建立了单体、二聚体和聚合物的分布作为解释相关专著中分子大小分布测试结果的基础。然而,具有较小粒度的现代色谱柱提供了更好的分辨率,并且还揭示了这里指定为低聚物的一类成分。本出版物针对以下问题解释了SEC Ig测试:-分子大小分布测试如何从使用最新的色谱技术中受益,而不会改变已建立的质量参数?是否有可能在数学上定义一种方法来解释具有相同分馏范围但分辨率不同的各种柱类型生成的色谱图?低聚物应如何考虑是否符合药典规范?
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引用次数: 0
An HPLC method to determine sennoside A and sennoside B in Sennae fructus and Sennae folium. 采用高效液相色谱法测定番泻子和番泻叶中番泻苷A和B的含量。
Q4 Medicine Pub Date : 2014-01-01
Immanuel Rosenthal, Evelyn Wolfram, Beat Meier

Introduction: The current Ph. Eur. monographs for senna pods, senna leaf and senna leaf dry extract standardised describe a photometric assay based on the Bornträger reaction to determine hydroxyanthracene glycosides, calculated as sennoside B. The method is timeconsuming, unspecific for sennosides and the precision is not adequate for a modern assay.

Aim: The photometric method shall therefore be replaced by a modern HPLC method. About 70 % of the total anthrachinone content in herbal drugs of senna species is due to sennoside A and sennoside B. These substances are therefore suitable for the standardisation of Senna products. The Japanese Pharmacopoeia (JP) already describes an HPLC method to determine sennoside A and sennoside B in the monograph for senna leaf. It uses ion-pair chromatography with tetraheptylammoniumbromide. The procedure described in the monograph has a runtime of 70 min.

Method: The adapted and validated method described here uses solid-phase extraction (SPE) which allows a selective sample preparation by using an anion exchange phase. A conventional RP C18 column Tosh TSKgel ODS-80TS (4.6 mm × 150 mm), 5 μm, was used as stationary phase and acetonitrile for chromatography R, water R, phosphoric acid R (200:800:1 V/V/V) as mobile phase. The flow rate was 1.2 mL/min, the column temperature 40 °C, the detection wavelength 380 nm, and the injection volume 20 μL. The runtime is 10 min, the chromatogram shows 2 peaks due to sennoside A/B and 2 additional smaller compounds. One of them is rhein-8-O-glucoside.

Results: The procedure has been successfully validated according to ICH guidelines. We analysed 6 batches of Senna. The pods (Senna angustifolia) showed a total content of sennoside A and B of 1.74-2.76 % m/m and the content of senna leaves was clearly lower with 1.07-1.19 % m/m, respectively.

Conclusion: The suggested method is considered to be suitable to determine sennoside A and sennoside B in senna leaves and senna pods. The consideration is based on the performed validation and on the results for the analysed samples. A short run time and better resolution are clear advantages of the suggested method, compared to other methods.

简介:目前博士学位。关于番泻叶豆荚、番泻叶和番泻叶干提取物标准化的专著描述了一种基于Bornträger反应的光度测定法,用于测定羟基蒽苷,计算为番泻叶苷b。该方法耗时,对番泻叶苷不特异性,精度不足以用于现代测定。目的:因此,光度法应被现代高效液相色谱法所取代。番泻泻属中草药中总蒽醌含量的70%左右是由于番泻泻甲和番泻泻乙。这些物质适合于番泻泻产品的标准化。日本药典(JP)已在番泻叶各论中描述了测定番泻叶皂苷A和B的高效液相色谱法。它使用离子对色谱法与四庚基溴化铵。本专著中描述的程序运行时间为70分钟。方法:本文描述的经过调整和验证的方法使用固相萃取(SPE),该方法允许使用阴离子交换相进行选择性样品制备。色谱柱为Tosh TSKgel ODS-80TS (4.6 mm × 150 mm), 5 μm,固定相为乙腈R,水R,磷酸R (200:800:1 V/V/V)为流动相。流速为1.2 mL/min,柱温40℃,检测波长380 nm,进样量20 μL。运行时间为10 min,色谱图显示2个峰,这是由于sennoside A/B和另外2个较小的化合物。其中之一是大黄-8- o -葡萄糖苷。结果:该方法已根据ICH指南成功验证。我们分析了6批塞纳。番泻豆荚中总皂苷a和B的含量为1.74 ~ 2.76% m/m,叶片中总皂苷含量较低,分别为1.07 ~ 1.19% m/m。结论:本方法适用于番泻泻叶和番泻泻豆荚中番泻泻皂苷A和B的含量测定。考虑是基于所执行的验证和分析样品的结果。与其他方法相比,所建议的方法具有运行时间短和分辨率高的明显优点。
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引用次数: 0
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Pharmeuropa bio & scientific notes
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