乙型脑炎减毒活疫苗生物学关键质量指标警戒限和行动限的建立与应用

 目的   建立适用于乙型脑炎减毒活疫苗（乙脑疫苗）生物学关键质量指标（critical quality attribution,CQA）的警戒限和行动限范围,开展趋势分析以及时发现异常趋势,指导疫苗生产。 方法   根据CQA的数据分布及特点,采用休哈特控制图法及阈值法建立限度范围,比较两种方法的结果,并采用适宜的限度范围对近3年的数据进行统计分析。 结果  乙脑疫苗的病毒滴度等生物学CQA数据为非正态分布,采用休哈特控制图建立的行动限范围有超出法规限的情况;而采用阈值法建立的行动限范围则均在法规限以内。采用阈值法建立的限度分析近3年乙脑疫苗各生产阶段生物学指标的状态,各指标超出行动限的点数均小于总点数的1%,表明乙脑疫苗各生产阶段处于稳定状态。 结论  阈值法为建立限度范围的适用方法;趋势分析中应结合数据本身特性与分布,选用适合的方法来建立限度标准,才能更为科学地将趋势分析工具用于指导实际生产。     

Application of different analytical methods for the characterization of non-spherical micro- and nanoparticles

Non-spherical micro- and nanoparticles have recently gained considerable attention due to their surprisingly different interaction with biological systems compared to their spherical counterparts, opening new opportunities for drug delivery and vaccination. Up till now, electron microscopy is the only method to quantitatively identify the critical quality attributes (CQAs) of non-spherical particles produced by film-stretching; namely size, morphology and the quality of non-spherical particles (degree of contamination with spherical ones). However, electron microscopy requires expensive instrumentation, demanding sample preparation and non-trivial image analysis. To circumvent these drawbacks, the ability of different particle analysis methods to quantitatively identify the CQA of spherical and non-spherical poly(1-phenylethene-1,2-diyl (polystyrene) particles over a wide size range (40 nm, 2 μm and 10 μm) was investigated. To this end, light obscuration, image-based analysis methods (Microflow imaging, MFI, and Vi-Cell XR Coulter Counter) and flow cytometry were used to study particles in the micron range, while asymmetric flow field fractionation (AF4) coupled to multi-angle laser scattering (MALS) and quasi elastic light scattering (QELS) was used for particles in the nanometer range, and all measurements were benchmarked against electron microscopy. Results show that MFI can reliably identify particle size and aspect ratios of the 10 μm particles, but not the 2 μm ones. Meanwhile, flow cytometry was able to differentiate between spherical and non-spherical 10 or 2 μm particles, and determine the amount of impurities in the sample. As for the nanoparticles, AF4 coupled to MALS and QELS allowed the measurement of the geometric (r_g) and hydrodynamic (r_h) radii of the particles, as well as their shape factors (r_g/r_h), confirming their morphology. While this study shows the utility of MFI, flow cytometry and AF4 for quantitative evaluation of the CQA of non-spherical particles over a wide size range, the limitations of the methods are discussed. The use of orthogonal characterization methods can provide a complete picture about the CQA of non-spherical particles over a wide size range.     

Sample size calculations for blinding assessment

Blinding is a critical component in randomized clinical trials along with treatment effect estimation and comparisons between the treatments. Various methods have been proposed for the statistical analyses of blinding-related data, but there is little guidance for determining the sample size for this type of data, especially if blinding assessment is done in pilot studies. In this paper, we try to fill this gap and provide simple methods to address sample size calculations for a “new” study with different research questions and scenarios. The proposed methods are framed in terms of estimation/precision or statistical testing to allow investigators to choose the best suited method for their goals. We illustrate the methods using worked examples with real data.     

Adaptive Design Space as an Integrated Component of Quality by Design

Introduction

The US Food and Drug Administration requires pharmaceutical companies to develop extensive process understanding prior to routine manufacturing of drug products. Through development and validation, drug manufacturers enhance their process understanding and identify an acceptable range of process parameters for each unit operation; this is referred to as the design space. Typically, limited work is done to study the effect of long-term raw material variations on the robustness of the design space. In the present study, the development of a design space for a tablet formulation containing two APIs (acetaminophen, caffeine) through a direct compression process was investigated.

Material and Methods

A design of experiment including different excipient ratios of microcrystalline cellulose and lactose, two croscarmellose sodium levels, four tablet compression forces, and four blend parameters was created using an industrial-size press to define a knowledge space. Quality attributes (disintegration time, dissolution, radial tensile strength, and friability) were measured and a design space derived. In order to test the robustness of the design space, raw material properties, specifically particle size of acetaminophen and ratio of lactose anhydrous to monohydrate, were modified. Also, compression parameters were varied.

Results

Tablets were analyzed for relevant critical quality attributes (CQAs) to investigate how variability in raw materials can change the design space. The modification of the process parameters was used as a means of compensating for raw material variability to produce tablets that met CQA requirements. An adaptive design space approach based on the adaptation of critical process parameters is proposed to facilitate the creation of tablets meeting specifications despite variation in raw material properties.     

Itraconazole suspension for intravenous injection: determination of the real component of complete refractive index for particle sizing by static light scattering

The purpose of this study is to assess the impact of real refractive indices, using different itraconazole suspensions, on the associated particle size distributions. Instrumental particle size measurement remains the practical option for determining the particle size distribution of a suspension. In this study, the suspension particle size distribution was measured by static light scattering, which requires knowledge of both the real and imaginary components of the complete refractive index. The real refractive indices of micronized itraconazole raw material, as well as vacuum-dried itraconazole suspension samples obtained from different formulations, polymorphs, manufacturing methods and particle size distributions, were determined using the Becke line method. Identical samples were analyzed by two contract laboratories in order to assess consistency. For the static light scattering equipment used in this study, the complete relative refractive index (RRI = n(particte) / n(dispersant) - ik) input required for software calculation is denoted by a refractive index kernel (RRI input) comprising a relative real component and an imaginary component. The reported real refractive indices for the itraconazole raw material as well as vacuum dried itraconazole suspension samples were different, ranging from 1.608 to 1.65 (selected kernel range of 120A010I to 124A010I). The imaginary component of itraconazole suspension was determined in a previous study to be 010I. The average real refractive index was calculated to be 1.62 (122A010I). The particle size distributions obtained using 120A010I and 124A010I were in good agreement with one generated using 122A010I. Therefore, itraconazole suspensions that were produced using different manufacturing methods/formulations or exhibited different particle size distributions/polymorphic forms may use 122A010I in determining particle size distribution. The particle size distributions determined using RRI input outside the range of 120A010I to 124A010I may not be reliable. However, it is recommended that similar investigations be conducted for other drug suspensions on a case-by-case basis.     

Characterisation of nanoparticulate systems by hydrodynamic chromatography

Particle size and particle size distribution can have a fundamental effect on the physical properties of colloidal dispersions. For many systems the measurement of average particle size is not sufficient, the presence of different size populations will have a strong influence on properties and could be related to the production process. Hydrodynamic chromatography (HDC) provides a method for the separation of polymers in solution or particles in suspension based on their size. In a packed column, the separation takes place in the inter-particle channels and the elution order is from large to small, analogous to gel permeation chromatography. The dynamic range of packed column HDC is from molecular size up to particles of greater than 1 microm. New instrumentation which can be used to determine the particle size distribution of a range of colloidal dispersions by packed column HDC is described. Data to support accuracy and precision of average particle size determination is presented as well as a number of case studies to illustrate the applicability of the technique to samples with polydisperse or multi-modal particle size distributions.     

An integrated Quality by Design (QbD) approach towards design space definition of a blending unit operation by Discrete Element Method (DEM) simulation

A combined Quality by Design (QbD) and Discrete Element Model (DEM) simulation-approach is presented to characterize a blending unit operation by evaluating the impact of formulation parameters and process variables on the blending quality and blending end point. Understanding the variability of both the API and the excipients, as well as their impact on the blending process are critical elements for blending QbD. In a first step, the QbD-methodology is systematically used to (1) establish the critical quality attribute content uniformity and to link this CQA to its surrogate blend homogeneity, (2) identify potentially critical input factors that may affect blending operation quality and (3) risk-rank these factors to define activities for process characterization. Subsequently, a DEM-simulation-based characterization of the blending process is performed. A statistical evaluation is finally presented, relating blend homogeneity of systems with low particle number to the regulatory requirements. Data are then used to map out a three-dimensional knowledge space, providing parameters to define a design space and set up an appropriate control strategy.     

Quantitative Microscopy: Particle Size/Shape Characterization,Addressing Common Errors Using ‘Analytics Continuum’ Approach

Particle size/shape characterization of active pharmaceutical ingredient (API) is integral to successful product development. It is more of a correlative property than a decision-making measure. Though microscopy is the only technique that provides a direct measure of particle properties, it is neglected for reasons like non-repeatability and non-reproducibility which is often attributed to a) fundamental error, b) segregation error, c) human error, d) sample randomness, e) sample representativeness etc. Using the “Sucrose” as model sample, we propose “analytics continuum” approach that integrates optical microscope PSD measurements complimented by NIR spectroscopy-based trending analysis as a prescreening tool to demonstrate sample randomness and representativeness. Furthermore, plethora of statistical tests are utilized to infer population statistics. Subsequently, an attribute-based control chart and bootstrap-based confidence interval was developed to monitor product performance. A flowchart to serve as an elementary guideline is developed, which is then extended to handle more complex situations involving API crystallized from two different solvent systems. The results show that the developed methodology can be utilized as a quantitative procedure to assess the suitability of API/excipients from different batches or from alternate vendors and can significantly help in understanding the differences between material even on a minor scale.     

药代动力学非房室模型终末段半衰期的算法研究

目的:评价非房室模型终末段半衰期(t1/2z)不同计算方法的准确度和精密度,为t1/2z算法优选提供依据.方法:在不同个体间变异(CV,CV＝15％,25％,35％)下,用静注二室房室模型参数进行数据模拟,分别获得1000个受试者完整时间与浓度(C-T)曲线数据,再按样本量(N,N=15,25,35,45)用Bootstrap方法抽样1000次,最后获得12个数据集,用4种方法(5点法,4点法,6点法,全点法)计算每个数据集消除相不同点时的t1/2z,其结果的均值与真值(静注二室模型的消除半衰期)比较作为准确度,其标准差大小作为稳定性.结果:当C-T曲线消除相有4个点时,5点法计算值最准确且较为稳定,而6点法最为稳定;当消除相3点时,4点法计算值准确度最高且较为稳定,而6点法最为稳定;消除相2点时,4种方法准确度稳定性皆低.结论:消除相有效浓度点应至少3点,5点法和4点法计算t1/2z较为合理.     

Development of a Design Space for a Unit Operation: Illustration Using Compression-Mix Blending Process for the Manufacture of a Tablet Dosage Form

This paper describes the development of an orthogonal design space for a compression-mix blending unit operation for the manufacture of tablet dosage form using an empirical approach. Potential critical process parameters identified through a risk assessment process were assessed through a full-factorial design of experiment for impact on material attributes and drug product critical quality attributes (DP CQA). The impact on each individual attribute measured as responses were subjected to statistical analysis by analysis of variance and regression models were built on the statistically significant effects (p < 0.05). Design space for relevant DP CQA was created using 95% predicted interval estimates. Orthogonal design space for the unit operation was proposed by overlaying design spaces generated for individual DP CQAs. The resulting orthogonal design space made implementation of manufacturing flexibility in to routine manufacturing process and into control strategy simpler and straightforward.     

Development of computerised procedures for the characterisation of the tableting properties with eccentric machines: extended Heckel analysis.

Heckel plots are a suitable and valuable method for analysis of powder compaction with very small amounts of powder. The determination is based upon a non-linear transformation of compression data and thus the signal errors that might be introduced into the analysis might be enlarged and become critical. The method of determination of true density affects the results dramatically as does the accuracy of the powder height determination. The porosity should be corrected for compression of the solid fraction. The accuracy of the powder height detection is the most demanding parameter. The statements are proven by simulations based on real data and analytic calculation. According to these highly corrected Heckel plots, the shape of the plots during the compression phase gives the information about fragmentation and plasticity and additionally about the time dependency of the compression behaviour within one compression on an eccentric press.     

Determination of sitagliptin in human urine and hemodialysate using turbulent flow online extraction and tandem mass spectrometry

High turbulence liquid chromatography (HTLC, or turbulent flow online extraction) and tandem mass spectrometry (MS/MS) methods for the determination of sitagliptin in human urine and hemodialysate were developed and validated to support clinical studies. A narrow bore large particle size reversed-phase column (Cyclone, 50 mm x 1.0 mm, 60 microm) and a BDS Hypersil C18 column (30 mm x 2.1 mm, 3 microm) were used as extraction and analytical columns, respectively. For the urine assay, the LLOQ was 0.1 microg/ml, the linear calibration range was 0.1 to 50 microg/ml, the interday precision (R.S.D.%, n=5) was 2.3-6.5%, and the accuracy was 96.9-106% of the nominal value. For the urine quality control samples (QCs), the intraday precision (R.S.D.%, n=5) and accuracy were 1.8-2.6% and 96.2-106% of the nominal value, respectively. The interday precision (R.S.D.%) for 56 sets of urine QCs over a 6-month period varied from 3.8% to 5.5% and the accuracy from 102% to 105% of the nominal value. For the hemodialysate assay, the LLOQ was 0.01 ng/ml, the linear dynamic range was 0.01-5.0 ng/ml, the interday precision was 1.6-4.1%, and the accuracy was 89.8-104% of the nominal value. For hemodialysate QCs, the intraday precision and accuracy varied from 2.3% to 8.9% and from 99.8% to 111% of the nominal value, respectively. These results demonstrated that both methods are selective, accurate, precise, reproducible, and suitable for quantifying sitagliptin in hemodialysate and human urine samples.     

Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.     

Reference material development for calibration and verification of image-based particle analyzers

The need is well recognized for suitable reference populations for calibrating and verifying the size and concentration accuracy of particle analysis instruments for use in the measurement of suspended protein particles in biopharmaceuticals. Polystyrene bead standards are normally used as a reference material for calibrating and validating particle analyzers. However, these standards, unlike protein particles, are easily detected and do not challenge the sensitivity of optical instruments. Groups of instruments verified only with beads can still exhibit significant differences in measuring concentrations of more challenging protein particles. To minimize these and obtain consistent concentration measurements between instruments, reference populations must closely resemble protein aggregates in possessing high transparency and a refractive index close to typical protein matrix fluids. This paper describes work on evaluating a promising reference candidate and the use of this to harmonize the performance of Micro-Flow Imaging instruments. Results show that use of a suitable reference population can significantly increase measurement consistency when multiple instruments are used to characterize the same protein particle suspension.     

Adaptive designs for comparative effectiveness research trials

Abstract

Medical and health policy decision-makers require improved design and analysis methods for comparative effectiveness research (CER) trials. In CER trials, there may be limited information to guide initial design choices. In general settings, adaptive designs (ADs) have effectively overcome limits on initial information. However, CER trials have fundamental differences from standard clinical trials including population heterogeneity and a vaguer concept of a “minimum clinically meaningful difference”. The objective of this article is to explore the use of a particular form of ADs for comparing treatments within the CER trial context. To achieve this, the authors review the current state of clinical CER. They also identify areas of CER as particularly strong candidates for application of novel AD and illustrate the potential usefulness of the designs and methods for two group comparisons. The authors found that ADs can stabilize power. Furthermore, the designs ensure adequate power for true effects are at least at clinically significant pre-planned effect size, or when variability is larger than expected. The designs allow for sample size savings when the true effect is larger or when variability is smaller than planned. The authors conclude that ADs in CER have great potential to allow trials to successfully and efficiently make important comparisons.     

中药药效研究方法进展

目的对近些年来国内外中药药效研究方法和思路进行综述 ,并与中药药理作用的特点相比较。方法查阅近些年来国内外公开发表有关研究论文 ,按中药药效研究方法分类汇总。结果与结论提出对中药药效研究方法的几点思考 :中药药效的实验研究应该重视药物对机体的整体协调作用 ;应集中兵力 ,尽最大可能复制出符合中医“证”的临床辨证标准的动物模型 ;中药药效的研究方法应该在阐明复方配伍的化学与生物学内涵上寻找突破口。     

Methodological Aspects of Acute Toxicity Testing Particularly LD50 Determinations Present use in Development of New Drugs

Abstract It is essential to have an adequate understanding of the acute toxic effects of a new drug. It may not be necessary however to blindly follow the full classical median lethal assay on every occasion. The LD50 was developed to obtain the maximum accuracy from a modest experimental size. The objective of the study and the limitations of the assay should not be overlooked however and undue precision should not be sought. Methodological aspects including species and maximum and minimum doses will be discussed. Other methods and approaches to acute toxicity will be recommended with a more broad approach to acute toxicity than lethality alone.     

Using Image Attributes to Assure Accurate Particle Size and Count Using Nanoparticle Tracking Analysis

Nanoparticle tracking analysis (NTA) obtains particle size by analysis of particle diffusion through a time series of micrographs and particle count by a count of imaged particles. The number of observed particles imaged is controlled by the scattering cross-section of the particles and by camera settings such as sensitivity and shutter speed. Appropriate camera settings are defined as those that image, track, and analyze a sufficient number of particles for statistical repeatability. Here, we test if image attributes, features captured within the image itself, can provide measurable guidelines to assess the accuracy for particle size and count measurements using NTA. The results show that particle sizing is a robust process independent of image attributes for model systems. However, particle count is sensitive to camera settings. Using open-source software analysis, it was found that a median pixel area, 4 pixels², results in a particle concentration within 20% of the expected value. The distribution of these illuminated pixel areas can also provide clues about the polydispersity of particle solutions prior to using a particle tracking analysis. Using the median pixel area serves as an operator-independent means to assess the quality of the NTA measurement for count.     

NonClinical Dose Formulation Analysis Method Validation and Sample Analysis

Nonclinical dose formulation analysis methods are used to confirm test article concentration and homogeneity in formulations and determine formulation stability in support of regulated nonclinical studies. There is currently no regulatory guidance for nonclinical dose formulation analysis method validation or sample analysis. Regulatory guidance for the validation of analytical procedures has been developed for drug product/formulation testing; however, verification of the formulation concentrations falls under the framework of GLP regulations (not GMP). The only current related regulatory guidance is the bioanalytical guidance for method validation. The fundamental parameters for bioanalysis and formulation analysis validations that overlap include: recovery, accuracy, precision, specificity, selectivity, carryover, sensitivity, and stability. Divergence in bioanalytical and drug product validations typically center around the acceptance criteria used. As the dose formulation samples are not true “unknowns”, the concept of quality control samples that cover the entire range of the standard curve serving as the indication for the confidence in the data generated from the “unknown” study samples may not always be necessary. Also, the standard bioanalytical acceptance criteria may not be directly applicable, especially when the determined concentration does not match the target concentration. This paper attempts to reconcile the different practices being performed in the community and to provide recommendations of best practices and proposed acceptance criteria for nonclinical dose formulation method validation and sample analysis.     

预算影响分析中药品市场份额的计算方法探讨

目的 探讨预算影响分析(BIA)中药品市场份额的计算方法,以期为BIA研究提供新思路.方法 针对部分药品具有多种适应证或药品说明书适应证不够明确且与报销目录适应证表述不一致的情况,从"人次数"这一概念出发,引入样本医院、每人次平均治疗时间等参数来进行市场份额计算;同时以肝硬化治疗药物为例演示计算过程.结果 与结论 以肝...