Process analytical technology (PAT) for biopharmaceutical products: Part II. Concepts and applications

Implementing real‐time product quality control meets one or both of the key goals outlined in FDA's PAT guidance: “variability is managed by the process” and “product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions.” The first part of the paper presented an overview of PAT concepts and applications in the areas of upstream and downstream processing. In this second part, we present principles and case studies to illustrate implementation of PAT for drug product manufacturing, rapid microbiology, and chemometrics. We further present our thoughts on how PAT will be applied to biotech processes going forward. The role of PAT as an enabling component of the Quality by Design framework is highlighted. Integration of PAT with the principles stated in the ICH Q8, Q9, and Q10 guidance documents is also discussed. Biotechnol. Bioeng. 2010; 105: 285–295. Published 2009 Wiley Periodicals, Inc.     

种子超干贮藏技术应用面临的问题和研究方向

种子超干贮藏技术对种质资源保存具有很大的应用前景,但鉴于目前的研究进展,其应用还存在诸多技术上的问题。本文对种子超干贮藏技术在种子库中应用所面临的挑战以及需要开展的研究内容进行了如下几个方面的讨论：种子贮藏安全含水量下限的多样性决定了超干技术应用的复杂性;超干贮藏含水量安全下限的确定尚未有可靠的方法;超干贮藏的种子吸胀前需要恰当的预处理以避免吸胀损伤;超干处理前外源抗氧剂预处理可能改善超干贮藏效果;无氧条件可能对超干贮藏具有增效作用;糖类物质以及两性物质对种子在超干处理及超干贮藏过程中的保护作用。     

Manufacturing and characterization of Pd nanoparticles formed on immobilized bacterial cells

AIMS: To fabricate and analyse Pd nanoparticles on immobilized bacterial cells. METHODS AND RESULTS: Biological ceramic composites (biocers) were used as a template to produce Pd(0) nanoparticles. The metal-binding cells of the uranium mining waste pile isolate, Bacillus sphaericus JG-A12 were used as a biological component of the biocers and immobilized by using sol-gel technology. Vegetative cells and surface-layer proteins of this strain are known to bind high amounts of Pd(II) that can be reduced to Pd(0) particles by the addition of a reducing agent. Sorption of Pd(II) by the biocers from a metal complex solution was studied by inductively coupled plasma mass spectroscopy analyses. After embedding into sol-gel ceramics, the cells retained their Pd(II)-binding capability. Pd(0) nanoclusters were produced by the addition of hydrogen as reducing agent after the sorption of Pd(II). The interactions of Pd(0) with the biocers and the formed Pd(0) nanoparticles were investigated by extended X-ray absorption fine structure spectroscopy. The particles had a size of 0.6-0.8 nm. CONCLUSIONS: Bacterial cells that were immobilized by embedding into sol-gel ceramics were used as a template to produce Pd nanoclusters of a size smaller than 1 nm. These particles possess interesting physical and chemical properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of embedded bacterial cells as template enabled the fabrication of immobilized Pd(0) nanoparticles. These particles are highly interesting for technical applications, such as the development of novel catalysts.     

Application of cell engineering technology to the tumour immunotherapeutic drug: a review

The concept of cell engineering is frequently mentioned in recent years. It's a comprehensive technology which is very powerful and useful. By using it, scientists can produce many kinds of proteins and other biological moleculars in large scale that are in great demand for the human being. Among these products, most can be used for tumor immunotherapy and have been confirmed to be very effective in tumour control or subsidiary treatment. Of them, most common and useful drugs include three sorts; tumour vaccines, cell factors and monoclonal antibodies. The paper will discuss in detail related drugs application advances in tumour immunotherapy.     

The use of thermal imaging in assessing skin temperature following cryotherapy: a review

Background

Cryotherapy is used in various clinical and sporting settings to reduce odema, decrease nerve conduction velocity, decrease tissue metabolism and to facilitate recovery after exercise induced muscle damage. The basic premise of cryotherapy is to cool tissue temperature and various modalities of cryotherapy such as whole body cryotherapy, cold spray, cryotherapy cuffs, frozen peas, cold water immersion, ice, and cold packs are currently being used to achieve this. However, despite its widespread use, little is known regarding the effectiveness of different cryotherapy modalities to reduce skin temperature.

Objectives

To provide a synopsis of the use of thermal imaging as a method of assessing skin temperature following cryotherapy and to report the magnitude of skin temperature reductions associated with various modalities of cooling.

Design

Structured narrative review.

Methods

Three electronic databases were searched using keywords and MESH headings related to the use of thermal imaging in the assessment of skin temperature following cryotherapy. A hand-search of reference lists and relevant journals and text books complemented the electronic search.

Summary

Nineteen studies met the inclusion criteria. A skin temperature reduction of 5–15 °C, in accordance with the recent PRICE (Protection, Rest, Ice, Compression and Elevation) guidelines, were achieved using cold air, ice massage, crushed ice, cryotherapy cuffs, ice pack, and cold water immersion. There is evidence supporting the use and effectiveness of thermal imaging in order to access skin temperature following the application of cryotherapy.

Conclusions

Thermal imaging is a safe and non-invasive method of collecting skin temperature. Although further research is required, in terms of structuring specific guidelines and protocols, thermal imaging appears to be an accurate and reliable method of collecting skin temperature data following cryotherapy. Currently there is ambiguity regarding the optimal skin temperature reductions in a medical or sporting setting. However, this review highlights the ability of several different modalities of cryotherapy to reduce skin temperature.     

Teaching about Scientific Origins: Taking Account of Creationism

The purpose of this study was to develop a model for measuring experimental design ability based on functional magnetic resonance imaging (fMRI) during biological inquiry. More specifically, the researchers developed an experimental design task that measures experimental design ability. Using the developed experimental design task, they measured both the paper experimental design ability and the fMRI experimental design ability of subjects. Subjects’ paper experimental design ability was measured using the quotient equation of experimental design ability, and their fMRI experimental design ability using the brain connectivity coefficient. According to the fMRI results, differences in design ability existed among subjects in terms of brain connectivity coefficient level during the experimental design task. The experimental design ability brain connectivity coefficient level and quotient for each subject were analysed. Statistically significant correlations between subjects’ connectivity strength level among brain activation regions and quotient value guided the establishment of a measuring model. The model measured experimental design ability and could predict an individual’s experimental design ability quotient using his or her brain connectivity coefficient. Hence, the model developed for this study for measuring experimental design ability based on fMRI may serve as a practical measurement of students’ scientific experimental design ability. Furthermore, this study could serve as a founding theory for measuring models of other scientific processing abilities such as observation, question generation, classification, hypothesis generation and hypothesis evaluation.     

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed^1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere^1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms^3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous^5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects^5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms^5,8-13.Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals^14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection^13,16,17 or stable transduction^5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells²⁰. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.     

Hybrid modeling framework for process analytical technology: application to Bordetella pertussis cultures

In the process analytical technology (PAT) initiative, the application of sensors technology and modeling methods is promoted. The emphasis is on Quality by Design, online monitoring, and closed-loop control with the general aim of building in product quality into manufacturing operations. As a result, online high-throughput process analyzers find increasing application and therewith high amounts of highly correlated data become available online. In this study, an hybrid chemometric/mathematical modeling method is adopted for data analysis, which is shown to be advantageous over the commonly used chemometric techniques in PAT applications. This methodology was applied to the analysis of process data of Bordetella pertussis cultivations, namely online data of near-infrared, (NIR), pH, temperature and dissolved oxygen, and off-line data of biomass, glutamate, and lactate concentrations. The hybrid model structure consisted of macroscopic material balance equations in which the specific reactions rates are modeled by nonlinear partial least square (PLS). This methodology revealed a significant higher statistical confidence in comparison to PLSs, translated in a reduction of mean squared prediction errors (e.g., individual root mean squared prediction errors calibration/validation obtained through the hybrid model for the concentrations of lactate: 0.8699/0.7190 mmol/L; glutamate: 0.6057/0.2917 mmol/L; and biomass: 0.0520/0.0283 OD; and obtained through the PLS model for the concentrations of lactate: 1.3549/1.0087 mmol/L; glutamate: 0.7628/0.3504 mmol/L; and biomass: 0.0949/0.0412 OD). Moreover, the analysis of loadings and scores in the hybrid approach revealed that process features can, as for PLS, be extracted by the hybrid method.     

Model-based analysis on the relationship of signal quality to real-time extraction of information in bioprocesses

Quality by design (QbD) is a current structured approach to design processes yielding a quality product. Knowledge and process understanding cannot be achieved without proper experimental data; hence requirements for measurement error and frequency of measurement of bioprocess variables have to be defined. In this contribution, a model-based approach is used to investigate impact factors on calculated rates to predict the obtainable information from real-time measurements (= signal quality). Measurement error, biological activity, and averaging window (= period of observation) were identified as biggest impact factors on signal quality. Moreover, signal quality has been set in context with a quantifiable measure using statistical error testing, which can be used as a benchmark for process analytics and exploitation of data. Results have been validated with data from an E. coli batch process. This approach is useful to get an idea which process dynamics can be observed with a given bioprocess setup and sampling strategy beforehand.