Intensification of a multi-product perfusion platform – managing growth characteristics at high cell density for maximized volumetric productivity

Integrated Continuous Biomanufacturing (ICB) provides many important strategic advantages for therapeutic protein production through process intensification, simplification and integration. Dramatic reductions in cost of goods manufactured can be achieved by pushing perfusion culture towards high productivity at moderate perfusion rate and integrating with multi-column capture. We have demonstrated that an in-house chemically defined medium designed for high volumetric productivity (VPR) can support clones producing different monoclonal antibodies in perfusion bioreactors at cell densities \u3e100 million viable cells/mL and VPR from 4 to 6 g/L/day. However, for other cell lines tested productivity could not be consistently sustained due to declining growth rate at high cell density. It was demonstrated that increased bleed rates could extend the culture duration for these clones but only with substantially lower cell density and productivity, and reverting to a less productive perfusion medium improved culture longevity to a certain degree. It was shown that continuous addition of a concentrated supplement to this medium could improve productivity to levels comparable to the high-VPR medium, but this appeared to be less effective for clones with lower specific productivity. Some clones producing the same biologic were observed to exhibit either sustained or declining growth rate at high cell density, indicating clonal variability should be considered as another factor that can affect this growth phenotype. Potential strategies to mitigate declining growth rate in high cell density perfusion culture will be discussed and additional case studies on the application of intensified perfusion will be examined

Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing

<p>Abstract</p> <p>Background</p> <p>High throughput sequencing (HTS) technologies have revolutionized the field of genomics by drastically reducing the cost of sequencing, making it feasible for individual labs to sequence or resequence plant genomes. Obtaining high quality, high molecular weight DNA from plants poses significant challenges due to the high copy number of chloroplast and mitochondrial DNA, as well as high levels of phenolic compounds and polysaccharides. Multiple methods have been used to isolate DNA from plants; the CTAB method is commonly used to isolate total cellular DNA from plants that contain nuclear DNA, as well as chloroplast and mitochondrial DNA. Alternatively, DNA can be isolated from nuclei to minimize chloroplast and mitochondrial DNA contamination.</p> <p>Results</p> <p>We describe optimized protocols for isolation of nuclear DNA from eight different plant species encompassing both monocot and eudicot species. These protocols use nuclei isolation to minimize chloroplast and mitochondrial DNA contamination. We also developed a protocol to determine the number of chloroplast and mitochondrial DNA copies relative to the nuclear DNA using quantitative real time PCR (qPCR). We compared DNA isolated from nuclei to total cellular DNA isolated with the CTAB method. As expected, DNA isolated from nuclei consistently yielded nuclear DNA with fewer chloroplast and mitochondrial DNA copies, as compared to the total cellular DNA prepared with the CTAB method. This protocol will allow for analysis of the quality and quantity of nuclear DNA before starting a plant whole genome sequencing or resequencing experiment.</p> <p>Conclusions</p> <p>Extracting high quality, high molecular weight nuclear DNA in plants has the potential to be a bottleneck in the era of whole genome sequencing and resequencing. The methods that are described here provide a framework for researchers to extract and quantify nuclear DNA in multiple types of plants.</p

Disruption of splicing-regulatory elements using CRISPR/Cas9 to rescue spinal muscular atrophy in human iPSCs and mice

We here report a genome-editing strategy to correct spinal muscular atrophy (SMA). Rather than directly targeting the pathogenic exonic mutations, our strategy employed Cas9 and guide-sgRNA for the targeted disruption of intronic splicing-regulatory elements. We disrupted intronic splicing silencers (ISSs, including ISS-N1 and ISS + 100) of survival motor neuron (SMN) 2, a key modifier gene of SMA, to enhance exon 7 inclusion and full-length SMN expression in SMA iPSCs. Survival of splicing-corrected iPSC-derived motor neurons was rescued with SMN restoration. Furthermore, co-injection of Cas9 mRNA from Streptococcus pyogenes (SpCas9) or Cas9 from Staphylococcus aureus (SaCas9) alongside their corresponding sgRNAs targeting ISS-N1 into zygotes rescued 56% and 100% of severe SMA transgenic mice (Smn , SMN2 ). The median survival of the resulting mice was extended to >400 days. Collectively, our study provides proof-of-principle for a new strategy to therapeutically intervene in SMA and other RNA-splicing-related diseases. -/- tg/

Assessment of Engineering Behavior and Water Resistance of Stabilized Waste Soils Used as Subgrade Filling Materials

Urban construction has generated substantial amounts of waste soils, impeding urban ecological development. With the aim of promoting waste recycling, waste soils possess a high potential for sustainable utilization in subgrade construction. However, these waste materials exhibit inadequate engineering properties and necessitate stabilization for an investigation into their long-term performance as subgrade filling materials. Initially, a thorough assessment and comparison were conducted to examine the key mechanical properties of lime- and cement-stabilized soils with mixed ratios (total stabilizer contents ranging from 2% to 8%). The results indicated that these soils met the requirements of subgrade materials except for the 2% lime-treated soil. Subsequently, to reveal the improvement in water resistance of stabilized waste soil (e.g., under conditions of rainfall or elevated groundwater table), the effects of soil densities and stabilizer contents on the disintegration characteristics were investigated using a range of disintegration tests. An evolutionary model for the disintegration ratio of stabilized soils was then developed to predict the process of disintegration breakage. This model facilitates the quantification of the lower disintegration rates and elevated disintegration time attributed to higher levels of compactness and stabilizer contents during a three-stage disintegration process. This enhances the understanding and evaluation of sustainable applications in stabilized waste soils used as subgrade filling materials

Multidimensional Parameter Estimation Method Based on Sparse Iteration in FDA-MIMO Radar

To accurately identify the range of each target, traditional Multiple-Input Multiple-Output (MIMO) radar techniques not only require designing a shift matrix to describe different range bins but also a large number of snapshots. To alleviate this problem, a multidimensional parameter estimation method based on sparse iteration is proposed for a MIMO radar with Frequency Diverse Array (FDA). The FDA-MIMO radar uses small frequency increments across the array elements, and its transmit steering vector is a function of both range and angle. On the basis of the feature of the FDA-MIMO radar, we consider a weighted lq (0<q ≤1) minimization problem that is solved using a sparse iterative algorithm. Finally, the target parameters (the amplitude, range, and angle) are obtained using a single snapshot. Moreover, numerical simulations are used to demonstrate the superior performance of the proposed method compared with those of DAS, IAA, and IAA-R