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.     

Rapid Parallel Adaptation to Anthropogenic Heavy Metal Pollution

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.     

Habitat temperature and the temporal scaling of cold hardening in the high Arctic collembolan, Hypogastrura tullbergi (Schäffer)

Abstract.  1. Cold tolerance is a fundamental adaptation of insects to high latitudes. Flexibility in the cold hardening process, in turn, provides a useful indicator of the extent to which polar insects can respond to spatial and temporal variability in habitat temperature.
2. A scaling approach was adopted to investigate flexibility in the cold tolerance of the high Arctic collembolan, Hypogastrura tullbergi , over different time-scales. The cold hardiness of animals was compared from diurnal warming and cooling phases in the field, and controlled acclimation and cooling treatments in the laboratory. Plasticity in acclimation responses was examined using three parameters: low temperature survival, cold shock survival, and supercooling points (SCPs).
3. Over time-scales of 24–48 h, both field animals from warm diurnal phases and laboratory cultures from a 'warm' acclimation regime (18 °C) consistently showed greater or equivalent cold hardiness to animals from cool diurnal phases and acclimation regimes (3 °C).
4. No significant evidence was found of low temperature acclimation after either hours or days of low temperature exposure. The cold hardiness of H. tullbergi remained 'seasonal' in character and mortality throughout was indicative of the summer state of acclimatization.
5. These data suggest that H. tullbergi employs an 'all or nothing' cryoprotective strategy, cold hardening at seasonal but not diel-temporal scales.
6. It is hypothesised that rapid cold hardening offers little advantage to these high Arctic arthropods because sub-zero habitat temperatures during the summer on West Spitsbergen are rare and behavioural migration into soil profiles offers sufficient buffering against low summer temperatures.     

A Review of Rapid Methods for the Analysis of Mycotoxins

An overview is presented of the analysis of mycotoxins by rapid methods such as: enzyme linked immuno-sorbent assay (ELISA); flow through membrane based immunoassay; immunochromatographic assay; fluorometric assay with immunoaffinity clean-up column or with a solid phase extraction clean-up column; and fluorescence polarization method. These methods are currently commercially available and are reliable, rapid methods. This review focuses on the basic principle of each rapid method as well as advantages and limitations of each method. Additionally, we address other emerging technologies of potential application in the analysis of mycotoxins.     

Molecular diagnosis and frequencies of primary hypolactasia in populations of Russia and neighboring countries

Normally, the ability to digest milk sugar (lactose) is present in every child, but not in every adult. The decrease in lactase synthesis (hypolactasia) results in the inability to digest whole milk. Recent studies of the Finnish population have associated lactase persistence in adults with allele T of the C/T_?13910 polymorphism located upstream of the lactase gene; a 100% correlation of primary hypolactasia with genotype C/C has been proved. In this study, the allele and genotype frequencies of C/T_?13910 were determined in populations of Russia. The frequencies of genotype C/C, varying from 36.6% in Russians to 88.2% in Chukchi, were close to the published medical and epidemiological data on the hypolactasia frequencies in these populations. Genotyping was carried out by three different methods to determine the optimal one. Genotype C/C proved to be the key determinant of primary hypolactasia. It was assumed that DNA diagnosis of genotype C/C provides a predictive test to detect primary hypolactasia long before its clinical manifestation.     

Isolation and functional characterization of fatty acid delta5-elongase gene from the liverwort Marchantia polymorpha L

Bryophyte Marchantia polymorpha L. produces C22 very-long-chain polyunsaturated fatty acid (VLCPUFA). Thus far, no enzyme that mediates elongation of C20 VLCPUFAs has been identified in land plants. Here, we report the isolation and characterization of the gene MpELO2, which encodes an ELO-like fatty acid elongase in M. polymorpha. Heterologous expression in yeast demonstrated that MpELO2 encodes delta5-elongase, which mediates elongation of arachidonic (20:4) and eicosapentaenoic acids (20:5). Phylogenetic and gene structural analysis indicated that the MpELO2 gene is closely related to bryophyte Delta6-elongase genes for C18 fatty acid elongation and diverged from them by local gene duplication.     

Leptospirosis y rickettsiosis,reto diagnóstico para el síndrome febril en zonas endémicas

This is the case of a 50-year-old male from the region of Urabá, Colombia, with a mixed infection by Rickettsia rickettsii and Leptospira interrogans serovar Copenhageni ST78 and negative test for malaria and dengue fever.The patient presented with febrile syndrome and was unresponsive to systemic antibiotic treatment, who finally died in the intensive care unit. We established the postmortem diagnosis through molecular typification of the two etiological agents. In the inspection at the patient’s home, we found a Rattus rattus specimen infected with L. interrogans of the same serovar found in him. We found no ticks parasitizing the domestic animals cohabitating with the patient.This case of a mixed infection with progressive and fatal symptoms in a patient with occupational risk in a tropical disease endemic zone highlights the importance of considering the potential presentation of simultaneous etiologies in patients with multiple medical visits for unresolved febrile syndromes associated with risky exposure during agricultural activities.     

DNA biosensors that reason

Despite the many designs of devices operating with the DNA strand displacement, surprisingly none is explicitly devoted to the implementation of logical deductions. The present article introduces a new model of biosensor device that uses nucleic acid strands to encode simple rules such as "IF DNA_strand(1) is present THEN disease(A)" or "IF DNA_strand(1) AND DNA_strand(2) are present THEN disease(B)". Taking advantage of the strand displacement operation, our model makes these simple rules interact with input signals (either DNA or any type of RNA) to generate an output signal (in the form of nucleotide strands). This output signal represents a diagnosis, which either can be measured using FRET techniques, cascaded as the input of another logical deduction with different rules, or even be a drug that is administered in response to a set of symptoms. The encoding introduces an implicit error cancellation mechanism, which increases the system scalability enabling longer inference cascades with a bounded and controllable signal-noise relation. It also allows the same rule to be used in forward inference or backward inference, providing the option of validly outputting negated propositions (e.g. "diagnosis A excluded"). The models presented in this paper can be used to implement smart logical DNA devices that perform genetic diagnosis in vitro.