In-line monitoring of protein concentration with MIR spectroscopy during UFDF

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance—Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.     

At-line raman spectroscopy and design of experiments for robust monitoring and control of miniature bioreactor cultures

The biopharmaceutical industry is moving toward a more quality by design (QbD) approach that seeks to increase product and process understanding and process control. Miniature bioreactor systems offer a high-throughput method enabling the assessment of numerous process variables in a controlled environment. However, the number of off/at-line samples that can be taken is restricted due to the small working volume of each vessel. This limitation may be resolved through the use of Raman spectroscopy due to its ability to obtain multianalyte data from small sample volumes fast. It can, however, be challenging to implement this technique for this application due to the complexity of the sample matrix and that analytes are often present in low concentration. Here, we present a design of experiments (DOE) approach to generate samples for calibrating robust multivariate predictive models measuring glucose, lactate, ammonium, viable cell concentration (VCC) and product concentration, for unclarified cell culture that improves the daily monitoring of each miniature bioreactor vessel. Furthermore, we demonstrate how the output of the glucose and VCC models can be used to control the glucose and main nutrient feed rate within miniature bioreactor cultures to within qualified critical limits set for larger scale vessels. The DOE approach used to generate the calibration sample set is shown to result in models more robust to process changes than by simply using samples taken from the “typical” process. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2740, 2019.     

Chaotic genetic patchiness in the pelagic teleost fish Sardina pilchardus across the Siculo-Tunisian Strait

ABSTRACT

This investigation aims at assessing patterns of spatial genetic structure of the teleost fish Sardina pilchardus across the Siculo-Tunisian Strait (a well-known discontinuous biogeographic area) and delineating putative genetic stocks within the species. For this purpose, a total of 180 specimens, collected from 11 locations stretching across the western and eastern Mediterranean coasts of Tunisia, were analysed genetically by means of 18 nuclear allozyme loci. The outcome of this study revealed strong genetic differentiation among populations, with the marked genetic distinctiveness of the central Tunisian population at Mahdia. Despite the delineation of seven well-defined genetic groups, no significant correlation was found between genetic and geographic distances. Besides, the recorded population subdivision did not align with biogeographic boundaries, suggesting the presence of chaotic genetic patchiness. Recent genetic bottlenecks were evidenced in S. pilchardus populations. Patchy migration patterns were recorded among the examined pairs of sardine populations. Among the recorded 16 polymorphic loci, GPI-2 and SOD appeared to be subject to natural selection. Patterns of population genetic differentiation and structuring were not found to be driven by outlier loci that appeared to be under selection. Furthermore, the detected neutral GPI-1 locus was found to be responsible for most of the genetic variation among identified genetic clusters. Hence, natural selection cannot cause the detected genetic heterogeneity among sardine samples. Different explanations to the origin of chaotic genetic patterns, observed within S. pilchardus, were discussed.     

Fragmented habitat compensates for the adverse effects of genetic bottleneck

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