Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Peter Grunwald in Pharmaceutical Biocatalysis, 2019
In the second example, the purification of MC is tackled by exploring the in vitro enzymatic action of Nb.BbvCI (Alves et al., 2016; Silva-Santos et al., 2019), an artificial nicking endonuclease derived from the natural, R.BbvCI type IIA restriction endonuclease (EC 3.1.21.4) of Bacillus brevis (Wei et al., 2016). Nb.BbvCI was engineered by mutating one of the catalytic subunits of the heterodimeric Rb.BbvCI (31 kDa + 32 kDa) in such a way that it recognizes a 7-base pair asymmetric sequence on DNA substrates and introduces a single cut in one of the DNA chains (50′-CCTCAGC-3′/GCTGAGG→CCTCAGC/GCˆTGAGG) (Heiter et al., 2005). At the early stage of vector design, a target site for Nb.BbvCI is strategically placed in the MP part of the PP backbone. The manufacturing process then starts with E. coli culture and recombination of PPs into target MC, followed by cell harvesting and alkaline lysis, precipitation with isopropanol and ammonium sulfate and diafiltration/concentration by microfiltration. In the subsequent in vitro digestion step, Nb.BbvCI cleaves one of the strands of the MPs and of non-recombined PPs. The introduction of a nick in these species modifies their topology from supercoiled (sc) to open circular (oc). Sc MCs, on the other hand, remain unaffected since they are devoid of the recognition site. Hydrophobic interaction or multimodal chromatography is finally used to purify sc MCs from MPs, oc MCs and oc PPs (Alves et al., 2016; Silva-Santos et al., 2019).
Distribution and Toxicity of Retroviral Vectors after Intracavitary Delivery in Mouse and Man
Eric Wickstrom in Clinical Trials of Genetic Therapy with Antisense DNA and DNA Vectors, 2020
The retroviral vector was prepared by transfecting PA317 cells with the XM6:anti-fos retroviral vector DNA, which had been purified by alkaline lysis and ultracentrifugation twice on CsCl gradients. Following transfection, the PA317 cells were split and then treated with G418 until individual clones could be identified and expanded. Each clone was then assayed for vector titer by analyzing the supernatant's ability to transfer G418 resistance to MCF-7 cells. The clones which had the highest titer of vector production were then frozen in numerous aliquots and tested for sterility, presence of replication-competent retroviral vector, and presence of mycoplasma (Cornetta et al., 1993). The media from the XM6:anti-fos retroviral producer clone passed the following quality control tests: MAP test (mouse antibody production), in vitro test for adventitious viruses, bacterial and fungal sterility testing, mycoplasma testing, southern blot analysis of the retroviral producer clone, ability to transfer vector DNA to other cells (analyzed by transfer of G418 resistance and by southern blotting of target cells to demonstrate efficient gene transfer). Because distribution is only meaningful if replication competent vector is not present, we performed a PG-4 S+L- assay and 3T3 amplification followed by a PG-4 S+L- assay. Cells were tested for replicative retroviral contamination both by co-culture of producer cells and by amplification of retroviruses in supernatants by culture with Mus dunni cells. These tests, which included appropriate controls, showed no detection of replication competent viruses.
Genetic analysis of the embryo
David K. Gardner, Ariel Weissman, Colin M. Howles, Zeev Shoham in Textbook of Assisted Reproductive Techniques, 2017
Lysis of the single embryonic cells and exposure of their genetic material to the PCR reagents is one of the most criti- cal steps, and greatly affects ADO rates and the efficiency and reliability of PGD (18). Among several options, the three most commonly used lysis solutions are water, alkaline lysis buffer, and proteinase K/sodium dodecyl sulfate (SDS) buf- fer. There is as yet no consensus as to which is superior. Water
The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia–haemoglobin E disease and chromosome balance using karyomapping
Published in Journal of Obstetrics and Gynaecology, 2022
Sirivipa Piyamongkol, Suchada Mongkolchaipak, Pimlak Charoenkwan, Rungthiwa Sirapat, Wanwisa Suriya, Tawiwan Pantasri, Theera Tongsong, Wirawit Piyamongkol
Biopsied cells were washed thoroughly in phosphate-buffered saline (PBS, Cell Signaling Technology, Theera Trading Co. Ltd., Bangkok, Thailand) with 0.1% polyvinyl alcohol (PVA, Sigma-Aldrich, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) before transference to microcentrifuge tubes. DNA extraction was performed using an alkaline lysis buffer protocol (Sermon et al. 1995). 2.5 μL of lysis buffer (0.75 μL of water, 1.25 μL of 0.1 M DTT and 0.5 μL of 1 M NaOH) was added, mixtures were incubated at 60 °C for 10 min. After that, a neutralisation buffer (2.5 μL of 0.4 M tricine) was added. Whole genome amplification with multiple displacement amplification (MDA, REPLI-g® Single Cell Kit, Chiangmai VM Co., Ltd., Chiang Mai, Thailand) was then carried out by manufacturer’s instructions. A mixture of 12.5 μL of water, 29 μL of reaction buffer and 1 μL of DNA polymerase (REPLI-g® Single Cell Kit) was added to extracted DNA, making a total volume of 50 μL. The mixtures were incubated at 30 °C for 2 h then at 65 °C for 5 min to inactivate the reaction.
Comet Assay analysis of DNA strand breaks after exposure to the DNA-incorporated Auger Electron Emitter Iodine-125
Published in International Journal of Radiation Biology, 2023
Marcus Unverricht-Yeboah, Kathrin Holtmann, Ralf Kriehuber
Triton X-100 (1:100) and DMSO (1:10) were freshly added to the alkaline lysis solution (2.5 M NaCl, 100 mM Na3EDTA, 10 mM Tris Base, pH 10). For cell lysis, the slides with embedded cells were submerged in the alkaline lysis solution and incubated for 20–24 h at 4 °C. The slides were washed twice with 4 °C cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na3EDTA) and then incubated for 40 min with alkaline electrophoresis buffer at 4 °C. Then, electrophoresis was done at 1 V/cm for 30 min on ice in fresh alkaline electrophoresis buffer. Slides were then rinsed for 10 min in neutralization buffer (0.4 M Tris Base, pH 7.5) and incubated for 5 min in 70% ethanol. After drying for 10–15 min at 37 °C, the DNA was stained with propidium iodide (PI, 0.5 µg/ml in distilled water) for 30 min in the dark at RT. After rinsing the slides gently with water and drying by 37 °C, the slides with the embedded lysed cells were analyzed.
Advances of droplet-based microfluidics in drug discovery
Published in Expert Opinion on Drug Discovery, 2020
Yuetong Wang, Zhuoyue Chen, Feika Bian, Luoran Shang, Kaixuan Zhu, Yuanjin Zhao
Reverse transcriptase PCR (RT-PCR), a technique augmenting the transcribed target RNA strands, has been widely applied to look into cell heterogeneity [91]. However, false-negative results may produce when PCR inhibitors in low-quality biological samples result in reduction of the amplification efficacy. To solve this drawback, Abate et al. resorted to a droplet-based microfluidic chip combining cell lysis and reagent addition for single-cell RT-PCR analysis. Within the droplets, cells were immersed into alkaline lysis buffer. Because of a high pH environment, cell nucleases and inhibitory proteins could be denatured. The droplets containing lysed-cell were then merged into droplets with PCR reagents. This more trustable and less time-consuming strategy offered improvements for single-cell RT-PCR analysis.
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