Rapid Sterility Test Systems in the Pharmaceutical Industry: Applying a Structured Approach to Their Evaluation, Validation and Global Implementation.

The current compendial sterility test has a 14-day incubation time and is often the time-limiting step in the Assess and Release Process of pharmaceutical products. There is an ever-increasing number of technologies available on the market that have benefits in addition to faster Time to Result, such as standardization and automation of readout (eliminating analyst subjectivity) and improved data integrity (including eliminating the need for contemporaneous verification of the result by another analyst). Regulators have been encouraging the pharmaceutical industry to adopt these innovative systems; however, it has taken a considerable time before receiving the first approvals from various health authorities (including both the European Medicines Agency and Food and Drug Administration) for the use of an alternative and rapid sterility test for the release of sterile drug product lots. This article describes a systematic 9-step approach to the evaluation, equipment qualification, validation, and deployment of alternative sterility tests that can be applied by pharmaceutical companies wanting to take advantage of the numerous benefits of alternative sterility tests. Two case studies are presented to illustrate the validation and implementation approach, including statistical methods. Although most of the steps toward implementation are aligned, the validation and transfer have been approached differently for each of the case studies because of differences in the chosen technology as well as independent company internal decisions to comply with validation guidelines. However, both case studies show successful implementation of an alternative sterility test for sterile drug products with an ∼50% reduced incubation time.

Non-inferiority testing for qualitative microbiological methods: Assessing and improving the approach in USP 1223.

The United States Pharmacopoeia (USP) presents two approaches for showing non-inferiority of an alternate qualitative microbiological method versus a compendial method. One approach compares the positive rates for the alternate and compendial methods at one spike level, while the other one compares multiple most probable number (MPN) estimates from a multi-spike design using a t-test. In this paper, we discuss these approaches under certain assumptions and propose a third approach that can be used for both single and multiple dilutions, which we call the generalized MPN (gMPN) approach. Simulations, using Poisson distributed numbers of microorganisms in test samples, confirm that the USP approach based on rates is not suitable, that the USP approach based on MPNs is appropriate for non-inferiority, but the gMPN approach outperforms the MPN-based approach and is therefore recommended.

Practical Applications of Biofluorescent Particle Counting in Environmental Monitoring Investigations.

Investigations into environmental monitoring (EM) excursions can be prolonged and do not always result in clear root causes or corrective and preventative actions. This article outlines how biofluorescent particle counting (BFPC) can be used in investigations to eliminate the inherent delays of culture-based methods. The application for investigations supplements routine EM, acting as a risk-reduction tool enabling real-time detection of viable microorganisms in air samples and supporting root cause analysis and remedial actions. The article includes guidance on how to use the technology, a real case study involving a mold excursion, and examples of business benefits achieved by various companies.

A comparison of spiking experiments to estimate the detection proportion of qualitative microbiological methods.

The detection proportion of a qualitative microbiological test method is the probability to detect a single micro-organism. A general expression for the moment estimator of the detection proportion is provided. It depends on the distribution of the spikes used in a validation study through its moment-generating function. Several forms of spiking experiments are compared on their estimation performance using simulations and assuming a generalized Poisson distribution (GPD) for the spikes. The optimal design, which minimizes the mean squared error of our proposed moment estimator, depends on the dispersion parameter of the GPD. The design that uses just one spiked solution instead of multiple solutions is optimal for Poisson and overdispersed Poisson and it is robust against distributions for the spikes.

Continuous Microbiological Environmental Monitoring for Process Understanding and Reduced Interventions in Aseptic Manufacturing.

This paper provides recommendations for quality oversight, manufacturing operations, and industry perspective of regulatory expectations to enable aseptic facilities to move toward real-time and continuous microbiological environmental monitoring, thereby reducing interventions and future replacement of Grade A settle plates and nonremote active air sampling. The replacement of traditional monitoring with biofluorescent particle-counting systems provides an improvement in process understanding and product safety and reduces operator manipulations, assuring product quality and real-time process verification. The future state pharmaceutical technology roadmaps include gloveless isolators with real-time and continuous monitoring for aseptic manufacturing.LAY ABSTRACT: This paper advocates the use of an alternative and relatively new method of monitoring the air for contamination in biopharmaceutical manufacturing facilities. The alternative method is based on a type of instrument the authors refer to as a biofluorescent particle counter (BFPC). The BFPC method has the advantage of being able to detect airborne microorganisms continuously and to record the actual time of detection. The replacement of traditional monitoring with BFPC systems can provide better data, which can be used to improve the understanding of contamination risks in complex manufacturing processes, ultimately providing more confidence in product safety. The authors present data showing the suitability of BFPC. This immediate result is very useful for picking up early any possible contamination and should, therefore, provide a better way to monitor and control the risk of contamination. As traditional monitoring methods require manual manipulation, an additional advantage of BFPC systems is that they can reduce manual manipulations. Elimination of all interventions is a goal in the industry, because although they are tightly controlled, interventions are an unwanted potential source of contamination.

Equivalence testing for similarity in bioassays using bioequivalence criteria on the relative bioactivity.

Similarity in bioassays means that the test preparation behaves as a dilution of the standard preparation with respect to their biological effect. Thus, similarity must be investigated to confirm this biological property. Historically, this was typically conducted with traditional hypothesis testing, but this has received substantial criticism. Failing to reject similarity does not imply that the 2 preparations are similar. Also, rejecting similarity when bioassay variability is small might simply demonstrate a nonrelevant deviation in similarity. To remedy these concerns, equivalence testing has been proposed as an alternative to traditional hypothesis testing, and it has found its way in the official guidelines. However, similarity has been discussed mainly in terms of the parameters in the dose-response curves of the standard and test preparations, but the consequences of nonsimilarity on the relative bioactivity have never been investigated. This article provides a general equivalence approach to evaluate similarity that is directly related to bioequivalence on the relative bioactivity of the standard and test preparations. Bioequivalence on the relative bioactivity can only be guaranteed for positive (only nonblanks) and finite dose intervals. The approach is demonstrated on 4 case studies in which we also show how to calculate a sample size and how to investigate the power of equivalence on similarity.

A Phase 2 Randomized Dose-Finding Study With Esmirtazapine in Patients With Primary Insomnia.

The antidepressant mirtazapine is an alternative to classical hypnotics, and this study investigated the efficacy and safety of esmirtazapine (Org 50081, the maleic acid salt of S-mirtazapine) in patients given a diagnosis of primary insomnia after acute (2-day) treatment. Patients aged 18 to 65 years with primary insomnia were randomized to receive placebo or 1.5-, 3.0-, or 4.5-mg esmirtazapine in a balanced 4-way crossover study; 2 sleep laboratory nights with polysomnography were separated by 5-day, single-blind placebo washout periods. Polysomnography-determined total sleep time (primary end point) and patient-reported total sleep time improved by at least 25 minutes with all 3 doses of esmirtazapine (P ≤ 0.001 vs placebo). Polysomnography-measured wake time after sleep onset (P ≤ 0.0001) and latency to persistent sleep also improved vs placebo (P ≤ 0.01, 3.0 and 4.5 mg). Patient-reported sleep quality improved with 3.0- and 4.5-mg esmirtazapine (P ≤ 0.01 and P ≤ 0.05, respectively, vs placebo). Morning alertness and contentment were not altered after esmirtazapine, and calmness increased with 4.5-mg esmirtazapine vs placebo. Evening questionnaires showed no difference in duration of daytime naps but reduced energy and ability to work/function after esmirtazapine treatment periods vs placebo (P < 0.05), although this effect was limited to the first night of each 2-night period. There were few adverse events, no serious adverse events, or clinically relevant treatment differences in vital signs, laboratory values, or electrocardiogram. Esmirtazapine doses of 1.5 to 4.5 mg/day significantly improved quantity and quality of sleep and were generally well tolerated, with no evidence of safety concerns or consistent pattern of residual effects.

Validation of qualitative microbiological test methods.

This paper considers a statistical model for the detection mechanism of qualitative microbiological test methods with a parameter for the detection proportion (the probability to detect a single organism) and a parameter for the false positive rate. It is demonstrated that the detection proportion and the bacterial density cannot be estimated separately, not even in a multiple dilution experiment. Only the product can be estimated, changing the interpretation of the most probable number estimator. The asymptotic power of the likelihood ratio statistic for comparing an alternative method with the compendial method, is optimal for a single dilution experiment. The bacterial density should either be close to two CFUs per test unit or equal to zero, depending on differences in the model parameters between the two test methods. The proposed strategy for method validation is to use these two dilutions and test for differences in the two model parameters, addressing the validation parameters specificity and accuracy. Robustness of these two parameters might still be required, but all other validation parameters can be omitted. A confidence interval-based approach for the ratio of the detection proportions for the two methods is recommended, since it is most informative and close to the power of the likelihood ratio test.

A comparison of test statistics for the recovery of rapid growth-based enumeration tests.

This paper considers five test statistics for comparing the recovery of a rapid growth-based enumeration test with respect to the compendial microbiological method using a specific nonserial dilution experiment. The finite sample distributions of these test statistics are unknown, because they are functions of correlated count data. A simulation study is conducted to investigate the type I and type II error rates. For a balanced experimental design, the likelihood ratio test and the main effects analysis of variance (ANOVA) test for microbiological methods demonstrated nominal values for the type I error rate and provided the highest power compared with a test on weighted averages and two other ANOVA tests. The likelihood ratio test is preferred because it can also be used for unbalanced designs. It is demonstrated that an increase in power can only be achieved by an increase in the spiked number of organisms used in the experiment. The power is surprisingly not affected by the number of dilutions or the number of test samples. A real case study is provided to illustrate the theory.

Is it possible to reduce the incidence of weekend oocyte retrievals in GnRH antagonist protocols?

A retrospective analysis of a large, randomized clinical trial (Engage) assessed whether adjusting the start day of ovarian stimulation and/or day of human chorionic gonadotrophin (HCG) trigger could minimize oocyte retrieval during weekends without adverse effects on clinical outcome. Patients received recombinant FSH/gonadotrophin-releasing hormone (GnRH) antagonist regimens, with stimulation starting on day 2 or 3 of menses. HCG was administered when at least three follicles of ⩾ 17 mm were present on ultrasound scan or 1 day later. The frequency distribution of the day of reaching the HCG criterion relative to stimulation initiation was analysed to determine the optimal stimulation start day (cycle day 2 or 3) depending on the weekday at which menses started, to minimize weekend retrieval. The number of oocytes retrieved and pregnancy rates were not affected by start day and/or delay in HCG administration in regularly ovulating women aged 18-36 years with bodyweight 60-90 kg, body mass index 18-32 kg/m(2) and menstrual cycle length 24-35 days. In recombinant FSH/GnRH antagonist regimens, it appears possible to minimize weekend oocyte retrieval by selecting the cycle day to initiate stimulation, day 2 when menses starts Friday-Tuesday, otherwise day 3 and if necessary in combination with a 1-day HCG delay. A retrospective analysis of the data collected from a large, randomized clinical trial (Engage) was conducted to assess whether adjusting the start day of ovarian stimulation and/or the day of human chorionic gonadotrophin (HCG) trigger could minimize the possibility of retrieving the mature egg cells during weekends, without an adverse effect on the clinical outcome. Patients were administered recombinant FSH/gonadotrophin-releasing hormone (GnRH) antagonist on either day 2 or 3 of menses in order to start ovarian stimulation. Ultrasound monitoring was subsequently performed to determine when at least three follicles of ⩾17 mm or more in diameter were present, and final maturation was induced by HCG administration either on that day or 1 day later, if necessary. The frequency distribution of day of reaching HCG criterion relative to ovarian stimulation initiation on cycle day 2 or 3 was analysed to determine the optimal ovarian stimulation day (day 2 or 3) depending on the weekday at which the menses started to minimize the possibility of weekend egg retrieval. Number of eggs retrieved and pregnancy rates were not found to be affected by day of stimulation and/or a 1-day delay in HCG administration. In a recombinant FSH/GnRH antagonist protocol, weekend egg retrieval can be minimized by selecting the appropriate cycle day to initiate ovarian stimulation (cycle day 2 when menses starts Friday to Tuesday, otherwise cycle day 3), and if necessary in combination with a 1-day HCG delay.

Comparative incidence of ovarian hyperstimulation syndrome following ovarian stimulation with corifollitropin alfa or recombinant FSH.

Corifollitropin alfa is a novel recombinant gonadotrophin with sustained follicle-stimulating activity. A single injection can replace seven daily injections of recombinant follicle-stimulating hormone (rFSH) during the first week of ovarian stimulation. All cases of ovarian hyperstimulation syndrome (OHSS) with corifollitropin alfa intervention in a gonadotrophin-releasing hormone antagonist protocol have been assessed in three large trials: Engage, Ensure and Trust. Overall, 1705 patients received corifollitropin alfa and 5.6% experienced mild, moderate or severe OHSS. In the randomized controlled trials, Engage and Ensure, the pooled incidence of OHSS with corifollitropin alfa was 6.9% (71/1023 patients) compared with 6.0% (53/880 patients) in the rFSH group. Adjusted for trial, the odds ratio for OHSS was 1.18 (95% CI 0.81-1.71) indicating that the risk of OHSS for corifollitropin alfa was similar to that for rFSH. The incidence of mild, moderate and severe OHSS was 3.0%, 2.2% and 1.8%, respectively, with corifollitropin alfa, with 1.9% requiring hospitalization, and 3.5%, 1.3% and 1.3%, respectively, in the rFSH arms, with 0.9% requiring hospitalization. Despite a higher ovarian response with corifollitropin alfa compared with rFSH for the first 7days of ovarian stimulation, the incidence of OHSS was similar. Corifollitropin alfa is a new agent used in ovarian stimulation treatment for IVF fertilization. One injection of corifollitropin alfa can replace seven injections of recombinant FSH (rFSH). In three studies of corifollitropin alfa treatment, we assessed all cases of ovarian hyperstimulation syndrome (OHSS), a potentially serious complication of ovarian stimulation treatment. Overall, 5.6% of the patients (95/1701) experienced OHSS. Two of the trials compared corifollitropin alfa versus rFSH. Because OHSS is relatively rare, we pooled the results of these trials to give a more reliable estimate of the incidence of OHSS. In the pooled analysis, 6.9% (71/1023) of patients receiving corifollitropin alfa had signs or symptoms of OHSS, compared with 6.0% in the rFSH group (53/880). The risk of OHSS with corifollitropin alfa treatment was similar to the risk of OHSS in patients who received rFSH: the incidence of mild, moderate and severe OHSS was 3.0%, 2.2% and 1.8%, respectively, in patients in the corifollitropin alfa treatment groups, with 1.9% requiring hospitalisation, and 3.5%, 1.3% and 1.3%, respectively, in patients in the rFSH treatment groups, with 0.9% requiring hospitalization. Although the ovaries respond more to corifollitropin alfa than to rFSH for the first 7days of ovarian stimulation, neither treatment regimen was significantly more likely to cause OHSS.