Comparison of different methods for decision-making in bioequivalence assessment

If the regulatory requirements are symmetrical, the use of symmetrical confidence intervals as a decision rule for bioequivalence assessment leads, as shown by simulations, to better level properties and an inferior power compared to a rule based on shortest confidence intervals. A choice between these two approaches will have to depend on a loss function. For asymmetric regulatory requirements, symmetrical confidence intervals should not be used; however, a decision can still be based on posterior probabilities, pr (theta epsilon [r1, r2]/x), or shortest confidence intervals. For purposes of inference, presentation and interpretation of results, we think that the use of symmetrical confidence intervals alone can be misleading and we therefore recommend that the posterior probabilities and densities, or at least the shortest confidence intervals, be given.     

High-throughput and automation advances for accelerating single-cell cloning,monoclonality and early phase clone screening steps in mammalian cell line development for biologics production

Mammalian cell line development is a multistep process wherein timelines for developing clonal cells to be used as manufacturing cell lines for biologics production can commonly extend to 9 months when no automation or modern molecular technologies are involved in the workflow. Steps in the cell line development workflow involving single-cell cloning, monoclonality assurance, productivity and stability screening are labor, time and resource intensive when performed manually. Introduction of automation and miniaturization in these steps has reduced the required manual labor, shortened timelines from months to weeks, and decreased the resources needed to develop manufacturing cell lines. This review summarizes the advances, benefits, comparisons and shortcomings of different automation platforms available in the market for rapid isolation of desired clonal cell lines for biologics production.     

Genome-wide analysis of single nucleotide variants allows for robust and accurate assessment of clonal derivation in cell lines used to produce biologics

A clonally derived (or “monoclonal”) cell line is a cell population derived from a single progenitor cell. Clonally derived cell lines are required for many biotechnological applications. For instance, recombinant mammalian cells used to produce therapeutic proteins are expected by regulatory authorities to be clonally derived. Assurance of clonal derivation (or “clonality”) is usually obtained from the characterization of the procedure used for cell cloning, for instance by assessing the success rate of single-cell sorting but not by assessing the cell line itself. We have developed a method to assess clonal derivation directly from the genetic makeup of cells. The genomic test of clonality is based on whole-genome sequencing and statistical analysis of single nucleotide variants. This approach quantifies the clonal fractions present in nonclonal samples and it provides a measure of the probability that a cell line is derived from a single cell. Upon experimental validation of the test, we show that it is highly accurate and that it can robustly detect minor clonal fractions of as little as 1% of the cell population. Moreover, we find that it is applicable to various cell line development protocols. This approach can simplify development protocols and shorten timelines while ensuring clonal derivation with high confidence.     

Retrospective assessment of clonal origin of cell lines

Cell lines used for the manufacture of recombinant proteins are expected to arise from a single cell as a control strategy to limit variability and ensure consistent protein production. Health authorities require a minimum of two rounds of limiting dilution cloning or its equivalent to meet the requirement of single cell origin. However, many legacy cell lines may not have been generated with process meeting this criteria potentially impeding the path to commercialization. A general monoclonality assessment strategy was developed based on using the site of plasmid integration for a cell's identity. By comparing the identities of subclones from a master cell bank (MCB) to each other and that of the MCB, a probability of monoclonality was established. Two technologies were used for cell identity, Southern blot and a PCR assay based on plasmid-genome junction sequences identified by splinkerette PCR. Southern blot analysis revealed that subclones may have banding patterns that differ from each other and yet indicate monoclonal origin. Splinkerette PCR identifies cellular sequence flanking the point(s) of plasmid integration. The two assays together provide complimentary data for cell identity that enables proper monoclonality assessment and establishes that the three legacy cell lines investigated are all of clonal origin.     

Monitoring occurrence,extinction, and colonization probabilities for gibbon populations

All gibbon species (Family: Hylobatidae) are considered threatened with extinction and recognized on the International Union for Conservation of Nature Red List of Threatened Species. Because gibbons are one of the most threatened families of primates, monitoring their status is now critically important. Long-term monitoring programs applying occupancy approaches, in addition to assessing occurrence probability, improves understanding of other population parameters such as site extinction or colonization probabilities, which elucidate temporal and spatial changes and are therefore important for guiding conservation efforts. In this study, we used multiple season occupancy models to monitor occurrence, extinction, and colonization probabilities for northern yellow-cheeked crested gibbon Nomascus annamensis in three adjacent protected areas in the Central Annamites mountain range, Vietnam. We collected data at 30 listening posts in 2012, 2014, and 2016 using the auditory point count method. Occurrence probabilities were highest in 2012 (0.74, confidence interval [CI]: 0.56–0.87) but slightly lower in 2014 (0.66, CI: 0.51–0.79) and 2016 (0.67, CI: 0.49–0.81). Extinction probabilities during the 2012–2014 and 2014–2016 intervals were 0.26 (0.14–0.44) and 0.25 (0.12–0.44), respectively. Colonization probabilities during 2012–2014 were 0.44 (0.19–0.73) and between 2014 and 2016 was 0.51 (0.26–0.75). Although local site extinctions have occurred, high recolonization probability helped to replenish the unoccupied sites and kept the occurrence probability stable. Long-term monitoring programs which use occurrence probability alone might not fully reveal the true dynamics of gibbon populations. We strongly recommend including multiple season occupancy models to monitor occurrence, extinction, and colonization probabilities in long-term gibbon monitoring programs.     

A photographic ageing technique used on warthog

The ratios of tusk-length: snout-width from a series of warthog (Phacochoerus aethiopicus) photographs were calculated and plotted against the date the photographs were taken. Regression lines, as well as the 95% confidence intervals, were calculated from the photographs and plotted. These regression lines fell into four distinct groups, representing Age Classes II through V. The average degree of error in the tusk-length: snout-width ratio was 3-01 units, a figure well within the above 95% confidence intervals.     

Evaluation of exact and asymptotic interval estimators in logistic analysis of matched case-control studies.

We compare six methods for constructing confidence intervals for a single parameter in stratified logistic regression. Three of these are based on inversion of standard asymptotic tests--namely, the Wald, the score, and the likelihood ratio tests. The other three are based on the exact distribution of the sufficient statistic for the parameter of interest. These include the traditional exact method of constructing confidence intervals, and two others, the mid-P and mean-P methods, which are modifications of this procedure that aim at reducing the conservative bias of the exact method. Using efficient algorithms, the six methods are compared by determination of their exact coverage levels in a series of conditional sample spaces. An incident case-control study of lung cancer in women is used to further illustrate the differences among the various methods. Computation of coverage functions is seen as a useful graphical diagnostic tool for assessing the appropriateness of different methods. The mid-P and the score methods are seen to have better coverage properties than the other four.     

Assessing Conformance with Benford’s Law: Goodness-Of-Fit Tests and Simultaneous Confidence Intervals

Benford’s Law is a probability distribution for the first significant digits of numbers, for example, the first significant digits of the numbers 871 and 0.22 are 8 and 2 respectively. The law is particularly remarkable because many types of data are considered to be consistent with Benford’s Law and scientists and investigators have applied it in diverse areas, for example, diagnostic tests for mathematical models in Biology, Genomics, Neuroscience, image analysis and fraud detection. In this article we present and compare statistically sound methods for assessing conformance of data with Benford’s Law, including discrete versions of Cramér-von Mises (CvM) statistical tests and simultaneous confidence intervals. We demonstrate that the common use of many binomial confidence intervals leads to rejection of Benford too often for truly Benford data. Based on our investigation, we recommend that the CvM statistic Ud2, Pearson’s chi-square statistic and 100(1 − α)% Goodman’s simultaneous confidence intervals be computed when assessing conformance with Benford’s Law. Visual inspection of the data with simultaneous confidence intervals is useful for understanding departures from Benford and the influence of sample size.     

Confidence intervals for the mean of diagnostic test charge data containing zeros

In this paper, we consider the problem of interval estimation for the mean of diagnostic test charges. Diagnostic test charge data may contain zero values, and the nonzero values can often be modeled by a log-normal distribution. Under such a model, we propose three different interval estimation procedures: a percentile-t bootstrap interval based on sufficient statistics and two likelihood-based confidence intervals. For theoretical properties, we show that the two likelihood-based one-sided confidence intervals are only first-order accurate and that the bootstrap-based one-sided confidence interval is second-order accurate. For two-sided confidence intervals, all three proposed methods are second-order accurate. A simulation study in finite-sample sizes suggests all three proposed intervals outperform a widely used minimum variance unbiased estimator (MVUE)-based interval except for the case of one-sided lower end-point intervals when the skewness is very small. Among the proposed one-sided intervals, the bootstrap interval has the best coverage accuracy. For the two-sided intervals, when the sample size is small, the bootstrap method still yields the best coverage accuracy unless the skewness is very small, in which case the bias-corrected ML method has the best accuracy. When the sample size is large, all three proposed intervals have similar coverage accuracy. Finally, we analyze with the proposed methods one real example assessing diagnostic test charges among older adults with depression.     

Voluntary recording scheme reveals ongoing decline in the United Kingdom hazel dormouse Muscardinus avellanarius population

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Bioequivalence of generic aerosol bronchodilators: what are the issues?

I have attempted to address some critical issues relating to the introduction of generic aerosol bronchodilators in Canada. I approached Genpharm to obtain information on the data submitted to the HPB, including the number of subjects involved, but the company refused to divulge this information because it was concerned about the use of such information by its competitors. In addition to the in-vitro testing conducted by the HPB, should a single pharmacodynamic study be sufficient to demonstrate the safety and efficacy of a drug that serves such a critical role in the prevention of serious illness and possibly death? If so, what will constitute the minimum requirements for the design of such a study? In general, what should be the minimum standards required for safety, efficacy and bioequivalence of aerosol bronchodilators? The next phase rests with the provincial governments. What criteria will they use to determine whether a generic aerosol bronchodilator will be considered bioequivalent? It is essential that the criteria for bioequivalence be developed by experts, and ideally those criteria should be agreed upon and accepted by federal and provincial regulatory bodies before a product is given the status of bioequivalence. Unless such a step is taken it will be difficult to have confidence that products can be considered interchangeable. The issue of interchangeability of aerosol bronchodilators demands immediate attention. Regulatory agencies are caught between those groups with vested interests on both sides. Since patients will either benefit or suffer as a consequence of regulatory decisions, action must be taken to ensure that the best decisions are made. Scientists, clinicians and government officials should convene as soon as possible to formulate a satisfactory approach to this problem of interchangeability. The medical and pharmaceutical professions need reliable information, and patients should not be denied less expensive generic drugs if it can be determined that they are comparable to the innovator''s product.     

Estimation and Confidence Intervals for Two-Stage Sample-Size-Flexible Design with LSW Likelihood Approach

Adaptive sample size methods have been a popular topic in the field of clinical trials. There are a few basic requirements for the adaptive methods to be acceptable from the international regulatory viewpoint. All valid methods need to control the overall type-I error rate at the pre-specified level. The rule of the interim and final decisions needs to be explicit and clearly documentable. It is extremely desirable that the method employed also provides estimation of the treatment effect in addition to the significance test. In this paper we describe the point and confidence interval estimation for the likelihood approach of sample-size adaptive design proposed by Li et al. (Biostatistics 3:277–287, 2002, J. Biopharm. Stat. 15:707–718, 2005). We use the median unbiased estimator (Cox and Hinkley, Theoretical Statistics, p. 273, 1974) for estimating the treatment effect and demonstrate that the estimator has small mean squared error compared to the naïve method, and that the confidence interval estimation has correct coverage probability.     

Mini-computer programs for bioequivalence testing of pharmaceutical drug formulations in two-way cross-over studies. Including a survey of current parametric evaluation techniques

For bioequivalence testing of pharmaceutical formulations of the same drug entity, it is not sufficient to carry out an analysis of variance on the characteristic to be evaluated (e.g., area under the plasma level vs time curve, half-life of elimination, time to plasma-peak level, plasma peak level) and to establish 'classical' 95% confidence intervals for the difference or the ratio of the characteristic concerned. In the past 10 years, several approaches have been proposed as an aid in decision-making: Westlake's 95% intervals, Rodda and Davis' probabilities, Fluehler's posterior probability histograms and the evaluation of the residual variation coefficient. A survey of these approaches is given, together with a discussion of their merits, their differences and their similarities. It is recommended that the final evaluation should be supported by probability density plots, which facilitate easy understanding of the differences and similarities between the various approaches. A bioequivalence study with two types of oral tablets containing bepridil, a new anti-anginal drug, is used as an example. Computer programs are presented, which enable the user to easily apply the various approaches in order to meet requirements of regulatory agencies.     

A multidisciplinary approach directed towards the commercial release of transgenic herbicide-tolerant rice in Costa Rica

This review discusses a multidisciplinary and multicomponent approach leading to the development and commercial release of transgenic Costa Rican rice varieties tolerant to the herbicide gluphosinate ammonium. We describe the field evaluations of the transgenic lines and their potential environmental impact, focusing on gene flow, particularly in relation to native wild Oryza species and weedy rice, based on trials performed in compliance with the national regulatory requirements of the country. We also present a socio-economic analysis of rice production in Costa Rica and the economic benefits of genetically modified (GM) rice as well as an environmental risk-benefit analysis for the deployment of GM rice. Additionally, food safety evaluation, intellectual property management, requirements for deregulation, and options for the commercialization of the new varieties are discussed. We also present results from a national survey aimed at assessing the level of support for GM crops in Costa Rica as this forms an integral component of our approach. Taken together, our results demonstrate that the adoption of these genetically improved rice varieties will provide clear benefits to Costa Rican rice growers and consumers.     

Confidence Intervals for Population Allele Frequencies: The General Case of Sampling from a Finite Diploid Population of Any Size

The estimation of population allele frequencies using sample data forms a central component of studies in population genetics. These estimates can be used to test hypotheses on the evolutionary processes governing changes in genetic variation among populations. However, existing studies frequently do not account for sampling uncertainty in these estimates, thus compromising their utility. Incorporation of this uncertainty has been hindered by the lack of a method for constructing confidence intervals containing the population allele frequencies, for the general case of sampling from a finite diploid population of any size. In this study, we address this important knowledge gap by presenting a rigorous mathematical method to construct such confidence intervals. For a range of scenarios, the method is used to demonstrate that for a particular allele, in order to obtain accurate estimates within 0.05 of the population allele frequency with high probability (%), a sample size of is often required. This analysis is augmented by an application of the method to empirical sample allele frequency data for two populations of the checkerspot butterfly (Melitaea cinxia L.), occupying meadows in Finland. For each population, the method is used to derive % confidence intervals for the population frequencies of three alleles. These intervals are then used to construct two joint % confidence regions, one for the set of three frequencies for each population. These regions are then used to derive a % confidence interval for Jost''s D, a measure of genetic differentiation between the two populations. Overall, the results demonstrate the practical utility of the method with respect to informing sampling design and accounting for sampling uncertainty in studies of population genetics, important for scientific hypothesis-testing and also for risk-based natural resource management.     

An Improvement of the Westlake Symmetric Confidence Interval

A common statistical method for assessing bioequivalence of two formulations of a chemical substance is the symmetric confidence interval of WESTLAKE (1972). As mentioned by WEST -LAKE (1981) and SCHUIRMAN (1981) a more powerful method consists of two one-sided t-tests. An (1-α)-confidence interval consistent with the two one-sided t-tests procedure is given by [min(α, 0), max (0, b)] where [a, b] is the conventional (1–2α)-confidence interval of the t-test. This “central” confidence interval is always a strict subset of the symmetric confidence interval and thus has more power in proving bioequivalence. The central confidence interval has properties comparable with those of the conventional one-sided confidence intervals.     

Length–weight relationships for three fish species from the middle and lower reaches of the Jinsha River,China

This paper provides the length–weight relationships of three fish species from the middle and lower reaches of the Jinsha River, China. Samples were obtained seasonally during 2013~2016 using fishing gear (drift gillnets and di long) and electroshock fishing techniques (backpack electrofishing control unit powered by 12 V). For each species, the sample size, length range, weight range, LWR, 95% confidence intervals of a and b, and coefficient of correlation were determined. Length–weight relationship (LWR) for one species was unknown to FishBase and new maximum lengths were recorded for two species.     

Reintroduced Eurasian beavers (<Emphasis Type="Italic">Castor fiber</Emphasis>): colonization and range expansion across human-dominated landscapes

The protected Eurasian beaver Castor fiber is recolonizing its former range hereby entering human-dominated landscapes. This ecosystem engineer can cause considerable damage to human infrastructures and agriculture, by feeding, digging and damming. To prevent human–wildlife conflict and ensure continued support from the local residents, a better understanding of habitat selection is required. By using species distribution models (SDMs) to quantify habitat requirements in our study area in Flanders, Belgium, based on 1792 occurrence data from 71 territories, and a fine-scale land use and vegetation map, we explored the potential for future beaver settlements. The results indicate that even in a highly human-dominated landscape, there is sufficient habitat available to support beaver populations. We highlight the importance of distance to water, willow stands, wetland vegetation and poplar trees. We show that there is currently sufficient habitat to support 924 territories (619–1515, 90% confidence interval) in Flanders (but this does not imply these locations are conflict-free). Our findings indicate that 12 year after the reintroduction, there continues to be a large expansion potential, both in range and in densities within the currently recolonized area. Our results can be used as a management tool in order to evaluate possible risks linked with the return of beavers in a human dominated landscape. At these critical locations, increased monitoring or structural measures can prevent conflicts. By preventing or quickly resolving human wildlife conflicts, long-term coexistence between humans and beavers can be achieved.