Best practices during bioanalytical method validation for the characterization of assay reagents and the evaluation of analyte stability in assay standards, quality controls, and study samples

Characterization of the stability of analytes in biological samples collected during clinical studies together with that of critical assay reagents, including analyte stock solutions, is recognized as an important component of bioanalytical assay validation. Deficiencies in these areas often come to light during regulatory inspections. Best practices, based on current regulatory guidance, for the assessment of these issues as they pertain to ligand binding and chromatographic assays are covered in this review. Additionally, consensus recommendations reached during the recent AAPS/FDA Workshop on bioanalytical assay validation are highlighted.     

Key elements of bioanalytical method validation for macromolecules

The Third American Association of Pharmaceutical Scientists/US Food and Drug Administration (FDA) Bioanalytical Workshop, which was held May 1 and 2, 2006, in Arlington, VA, addressed bioanalytical assays that are being used for the quantification of therapeutic candidates in support of pharmacokinetic evaluations. One of the main goals of this workshop was to discuss best practices used in bioanalysis regardless of the size of the therapeutic candidates. Since the last bioanalytical workshop, technological advancements in the field and in the statistical understanding of the validation issues have generated a variety of interpretations to clarify and understand the practicality of using the current FDA guidance for assaying macromolecular therapeutics. This article addresses some of the key elements that are essential to the validation of macromolecular therapeutics using ligand binding assays. Because of the nature of ligand binding assays, attempts have been made within the scientific community to use statistical approaches to interpret the acceptance criteria that are aligned with the prestudy validation and in-study validation (sample analysis) processes. We discuss, among other topics, using the total error criterion or confidence interval approaches for acceptance of assays and using anchor calibrators to fit the nonlinear regression models.     

FDA工艺验证指南的特点分析

有效的工艺验证对保证药品质量至关重要,2011年1月24日,FDA发布了工艺验证指南《Process Validation:General Principles and Practices》。该指南概况了工艺验证的一般原则和方法,将工艺验证与产品生命周期概念以及现有的FDA/ICH指南进行了整合,如Q8(R2)药品研发、Q9质量风险管理和Q10药品质量体系。从技术审评的角度,对FDA新版工艺验证指南的主要特点进行讨论分析。     

US FDA/EMA harmonization of their bioanalytical guidance/guideline and activities of the Global Bioanalytical Consortium

The 2011 annual conference of the American Association of Pharmaceutical Scientists, held in Washington DC, USA, hosted a roundtable entitled: 'Update of the US FDA/European Medicines Agency (EMA) harmonization of their bioanalytical guidance - Global Bioanalytical Consortium activity and impact on small and large molecules.' The roundtable was initiated with a presentation from CT Viswanathan on the history of the revision of the FDA guideline on bioanalytical method validation. It was followed by a presentation by Jan Welink who presented an update on the final European Medicines Agency guideline on bioanalytical method validation with relevance to ongoing harmonization efforts. The final presentation was by Fabio Garofolo on the progress of the Global Bioanalytical Consortium harmonization teams for small and large molecules. Brian Booth and Sam Haidar of the FDA updated the audience on the status of the revision of the FDA bioanalytical guidance. The roundtable was moderated by Stephen Lowes.     

Biomarkers to improve the benefit/risk balance for approved therapeutics: a US FDA perspective on personalized medicine

This article highlights a current US FDA perspective concerning the use of biomarker-based diagnostics for personalized medicine. Specifically, current biomarkers that have application for improving the benefit/risk profile of already approved drugs are discussed. The success of biomarkers for use in personalized medicine depends on many factors, including proper evaluation of the usefulness of the biomarker for assessing the event of interest, and the safety and effectiveness of the diagnostic device used to measure the biomarker, which includes appropriate analytical and clinical validation. These points along with the many regulatory concerns regarding co-labeling of drugs and devices and future aspects, such as co-development, will be discussed in this regulatory science focus.     

Capsule review on bioanalytical method transfer: opportunities and challenges for chromatographic methods

With the globalization of drug development activities, transferring a validated bioanalytical procedure to a different site within a pharmaceutical company, or to one or multiple contract research organizations has been dramatically increased in recent years. Undeniably, bioanalytical method transfer is the needed step prior to routine sample analysis at the receiving laboratory. It is clearly stated in the 2001 US FDA Guidance on Bioanalytical Method Validation that a partial validation is needed for method transfer between laboratories. In the current EMA draft guidelines on method validation, the necessity of a method transfer is also emphasized. However, the above guidelines do not give many details on how and when a method transfer validation should be conducted. There is a need for a step-by-step deliberation on the overall strategies, procedures and even technical details for a successful bioanalytical method transfer. In this article, we review the contemporary information available in the scientific literature on method transfer and illustrate various bioanalytical method transfer scenarios using case studies. A 'flexible and fit-for-purpose' bioanalytical method transfer strategy is proposed.     

Workshop Report: Crystal City VI—Bioanalytical Method Validation for Biomarkers

With the growing focus on translational research and the use of biomarkers to drive drug development and approvals, biomarkers have become a significant area of research within the pharmaceutical industry. However, until the US Food and Drug Administration’s (FDA) 2013 draft guidance on bioanalytical method validation included consideration of biomarker assays using LC-MS and LBA, those assays were created, validated, and used without standards of performance. This lack of expectations resulted in the FDA receiving data from assays of varying quality in support of efficacy and safety claims. The AAPS Crystal City VI (CC VI) Workshop in 2015 was held as the first forum for industry-FDA discussion around the general issues of biomarker measurements (e.g., endogenous levels) and specific technology strengths and weaknesses. The 2-day workshop served to develop a common understanding among the industrial scientific community of the issues around biomarkers, informed the FDA of the current state of the science, and will serve as a basis for further dialogue as experience with biomarkers expands with both groups.     

生物样品分析中的方法学验证

目的:介绍在色谱分析方法中方法学验证与质控的内容及其有待发展的方向。方法:参阅国际上有关方法学验证的指导原则、国家食品药品监督管理局管理规范,结合本实验室的标准操作规程,对方法学验证与质控的内容及存在的问题进行综合分析。结果:总结了方法学验证和质控的内容,及方法学验证中存在的可能的风险。结论:已建立的方法学验证和质控内容基本完善,但方法学验证的标准也应该根据实际情况制定。     

A comparison of the prediction accuracy of two IVIVC modelling techniques

The goal when developing an in vitro-in vivo correlation (IVIVC) model is the ability to accurately predict the in vivo plasma concentration profile of a drug formulation using only its in vitro dissolution data. The prediction accuracy of any model depends on the reliability of the method used to develop it. Some statistical concerns regarding methods based on deconvolution have been highlighted and a convolution based technique has been proposed as an alternative. This comparison shows, by means of a simulation study, that the modelling approach which uses convolution produces far more accurate results, accurately predicting the observed plasma concentration-time profile and, therefore, comfortably meeting the FDA validation criteria. The fact that the model developed using the deconvolution based technique fails to describe the simulated data and thus fails the FDA validation test when it ought to pass should be of great concern to those currently implementing this method.     

Development and validation of a preclinical food effect model

A preclinical canine model capable of predicting a compound's potential for a human food effect was developed. The beagle dog was chosen as the in vivo model. A validation set of compounds with known propensities for human food effect was studied. Several diets were considered including high-fat dog food and various quantities of the human FDA meal. The effect of pentagastrin pretreatment was also investigated. The high-fat dog food did not predict human food effect and was discontinued from further evaluation. The amount of FDA meal in the dog was important in the overall prediction of the magnitude of human food effect. Fed/fasted Cmax and AUC ratios using a 50-g aliquot of the FDA meal in the dog were in the closest qualitative agreement to human data. Pentagastrin pretreatment did not affect the AUC in the fed state, but increased the fasted AUC for weakly basic compounds. Pentagastrin pretreatment and a 50-g aliquot of the FDA meal in the dog predicted the human food effect for a validation set of compounds. This model, which is intended for compound screening, will be helpful for determining food effect as a liability when compounds progress from discovery to clinical development.     

药品中遗传毒性杂质的评估和控制

目的 综述药品遗传毒性杂质控制相关指南和法规,为制药企业执行国际标准和准则提供一些建议和思路。方法 通过查找数据库如Pubmed、Medline及欧洲药品管理局(European Medicines Agency,EMA)、美国食品药品监督管理局(US Food and Drug Administratio,U.S.FDA)、人用药品注册技术要求国际协调会议(ICH)等网站,比较各指南法规关于遗传毒性控制限度和控制措施的异同点,为遗传毒性杂质的控制提供一个可行性步骤。结果 通过比较发现,EMA、U.S.FDA和即将出版的ICH M7指南在关键原则的应用方面如毒理学关注阈值(threshold of toxicological concern,TTC)、风险评估步骤、杂质5分类法等基本相同,但现行EMA和U.S.FDA法规存在分歧,不利于其有效执行,而ICH M7将为遗传毒性杂质的控制提供一个可行框架。结论 目前还缺乏完善有效的遗传毒性控制指南,ICH M7将解决U.S.FDA 和EMA 指南间分歧,更好地指导制药企业遗传毒性杂质的控制。     

A contract research organization's response to the new FDA guidances for bioequivalence/bioavailability studies for orally administered drug products

The new FDA Guidance for Industry BA and BE Studies for Orally Administered Drug Products--General Considerations and Average, Population, and Individual Approaches to Establishing Bioequivalence imply significant changes in the areas of enrollment, cost, ethics, time, entry, validation applications (EVAs), and statistical and pharmacokinetic methods. The changes from three-period to two-period design for food effect studies, the elimination of most steady state studies, and the analyses of only the active moiety or ingredient are welcome. However, if the current guidances are adopted, additional time will be needed for participants, and more participants will be needed, resulting in higher costs to drug developers. The PK parameters needed to assess BE and the need for replicate designs for drugs with long t1/2 are still unclear. Finally, the advantages of the aggregate property of the FDA metric versus the disaggregate criteria are challenged, and four bioequivalence criteria are proposed.     

Comparison of FDA (2018) and EAEU Regulatory Requirements for Bioanalytical Method Validation

Pharmaceutical Chemistry Journal - US FDA requirements published in the new 2018 guidance for bioanalytical method validation and the necessity to confirm their reliability for determining analyte...     

Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays

Abstract The Third AAPS/FDA Bioanalytical Workshop, entitled “Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays” was held on May 1–3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached. The views expressed in this article are those of the authors and do not reflect official policy at the FDA. No official endorsement by the FDA is intended or should be inferred.     

Method transfer for ligand-binding assays: recommendations for best practice

To support clinical trials, bioanalytical methods are often transferred from one laboratory to another. With the rising number of large-molecule therapeutic proteins submitted for US FDA approval, the demand for large-molecule bioanalytical support and, subsequently, method transfer increases. Ligand-binding assays are the methods most commonly used to quantify endogenous and therapeutic proteins for the assessment of biomarkers and pharmacokinetic parameters. The goal of this review is to provide an overview of ligand-binding assay method transfer, essential parameters for partial method validation and to lay out a strategy to increase the chance of success. The recommendations herein are based on a summary of current publications and the authors' specific experiences, to help increase workload efficiency, maintain positive collaborations with partners and meet program timelines.     

Validated HPLC method for the determination of ranitidine in plasma

A validated HPLC method for the determination of ranitidine in human plasma is presented. Sulfanilamide as internal standard (IS) was used. Plasma samples were purified by solid phase extraction (SPE) using a copolymeric [poly(divinyl-benzene-co-N-vinylpyrrolidone)] column ("Oasis Waters"). Mobile phase consisting of dibasic potasium phosphate 0.08 M/acetonitrile/methanol/triethylamine 0.05% (89.5:3:7:0.05) pH5 was used at a flow rate of 0.9 ml/min on a C18 column (Nova-Pack, 3,9 x 300 mm, Waters). The eluate was monitored using an UVNis detector set at 300 nm. Ratio of peak area of ranitidine to sulfanilamide was used for the quantitation of plasma samples. FDA criteria for bioanalytical validation was used to validate the method. Linearity was assessed between 100-1600 ng/ml, the limit of quantitation was 100 ng/ml and recovery was greater than 94%. Accuracy, precision and selectivity met the current recommendations for bioanalytical method validation. The method was successfully used in a bioavailability study of a ranitidine tablet in healthy volunteers.     

Increased importance of the documented development stage in process validation

Current trends in pharmaceutical quality assurance moved when the Federal Drug Administration (FDA) of the USA published its new guideline on process validation in 2011. This guidance introduced the lifecycle approach of process validation. In this short communication some typical changes from the point of view of practice of API production are addressed in the light of inspection experiences. Some details are compared with the European regulations.     

Use of pilot plant facilities to aid validation programs

According to the FDA guidelines (1), pilot plant facilities may be used to generate material to support process validation studies. The guidelines also state that process modifications are an inevitable part of process development and scale-up. However, if such modifications are made, there is a need to demonstrate that the products from both the old and new processes are comparable (2). Both of these guidelines were combined and applied during a recent development of a product at Genentech. In order to implement the process modifications and demonstrate product comparability, the pilot plant facilities were used for these production runs. As the process had changed and the product was being prepared for BLA submission, in process samples from the new manufacturing process were also required to support the process validation studies. By using pilot plant facilities to implement the process modifications, test product comparability, and produce material for process validation, we were able to minimize the impact of such work on the large-scale manufacturing facility.     

非临床毒理学研究使用全切片图像进行组织病理学评价和同行评议的研究进展

经过验证遵从良好实验室规范（GLP）并适合目的的全切片图像（WSI）系统,不仅可用于GLP条件下非临床毒理学研究的数字组织病理学评价和同行评议,而且可提高全球化数字组织病理学评价和同行评议的灵活性,并可提供数字化图像数据用于今后人工智能和机器学习图像分析。简要介绍了美国食品和药品监督管理局和经济合作与发展组织关于在GLP条件下非临床毒理学研究中使用WSI或数字组织病理学的指导原则,硬件和软件的验证,合同研究组织验证WSI数据的采集和传输,委托方验证WSI数据的下载和查看,以及使用经过验证的WSI系统对大鼠吸入毒理学试验所开展的数字组织病理学评价和同行评议,以期为中国药物非临床毒理学研究使用WSI进行组织病理学评价和同行评议提供一定参考,加快与国际先进国家接轨的进程。     

A comprehensive approach to qualify and validate the essential parameters of an in vitro release test (IVRT) method for acyclovir cream, 5%

The rate of release of an active pharmaceutical ingredient (API) from a topical semisolid dosage form can be influenced by its physical and structural properties. An In Vitro Release Test (IVRT) is an established method to characterize this rate of API release and compare the underlying sameness in product quality characteristics. The purpose of this work was to validate an IVRT method to compare acyclovir cream, 5% products. However, despite widespread use of the IVRT since 1997, there has been no established approach to validate an IVRT method. Our approach included: 1) qualification of the diffusion cell apparatus, 2) qualification of the laboratory, 3) validation of the HPLC analytical method, and 4) validation of numerous critical parameters of the IVRT method, itself, and resulted in a comprehensive and successful IVRT method validation. Subsequent to the IVRT validation work described here, the U.S. Food and Drug Administration (FDA) drafted a guidance on the development and validation of an IVRT method for acyclovir cream, 5%. Although there are notable differences between our approach and the approach in that guidance, this report illustrates how many of the same essential qualification parameters and validation concepts were considered and systematically addressed in our approach to IVRT validation.