Acoustic Wave Separation – A non-filtration approach for continuous clarification of perfusion cell culture prior to capture chromatography

Advances in perfusion cell culture have led to cell densities in excess of 100 million cells/mL with product titers similar to those obtained in fed batch (3-5 g/L). This performance has necessitated improvements in the yield and efficiency of the cell harvest and clarification stage to generate a stream of Harvested Cell Culture Fluid (HCCF) for capture chromatography and subsequent downstream processing. This is further driven by the evolution of continuous processes where there is a preference for a continuous feed of HCCF available for direct load to the continuous multicolumn capture chromatography step. In the present work we report on a novel disruptive and scalable single-use technology for cell retention during perfusion cell culture based on an acoustophoretic separation. Acoustic Wave Separation (AWS) technology exploits the use of low frequency acoustic forces to generate a three-dimensional standing wave across a flow channel. Recirculating cell culture from a perfusion bioreactor enters the flow channel and passes below the acoustic zone. The product-containing stream of HCCF is removed from the recirculating cell culture by passage through the acoustic zone. This yields a well clarified HCCF that can be polished using a small area filter. We report the continuous cell retention during a perfusion culture of a CHO cell line expressing a mAb. At process development (PD) scale we demonstrate the ability to continuously process CHO cell culture and retain cells at densities of up to 100 million cells/mL, at flow rates of up to 2 bioreactor volumes per day. Since the clarification technology does not involve the use of hollow fiber tangential flow filtration (TFF) we ensure 100% transmission of the mAb through the AWS device. The closed system remains operational for up to 60 days enabling this scalable technology to be suitable for use in clinical manufacture. The post-AWS HCCF is 99% clarified and any residual cellular material can be removed using a small gamma stable membrane filter or directly loaded onto a 0.2 micron filter prior to chromatography. Additionally, no demonstrable adverse effects have been identified for the quality of the HCCF, the product itself, or the viability of the returning perfusion cell culture following cell retention using AWS technology. AWS technology enables the continuous cell retention from recirculating cell culture withdrawn from perfusion bioreactors in a single-use operation. AWS technology has been shown to perform well at cell densities of up to 100 million cells/mL, so is well positioned to meet the cell retention requirements of emerging higher cell density perfusion processes that are gaining momentum in the biotech space. This novel cell retention approach offers economic benefits in terms of yield improvement as well as eliminating the hollow fiber TFF operation. This offers the advantage of a stable mAb concentration in the HCCF stream during the perfusion process. This facilitates improved process control since the volume of HCCF to load on to the capture columns remains constant which is especially important during continuous multicolumn chromatography. By comparison with hollow fiber TFF, the mAb concentration varies during the cell retention process making an integrated process more complex to control

6-methylsalicylic acid polyketide synthetase-enzyme purification and gene cloning

Bibliography: p. 258-274

Pharmacologic Inhibition of Site 1 Protease Activity Inhibits Sterol Regulatory Element-Binding Protein Processing and Reduces Lipogenic Enzyme Gene Expression and Lipid Synthesis in Cultured Cells and Experimental Animals

Sterol regulatory element-binding proteins (SREBPs) are major transcriptional regulators of cholesterol, fatty acid, and glucose metabolism. Genetic disruption of SREBP activity reduces plasma and liver levels of cholesterol and triglycerides and insulin-stimulated lipogenesis, suggesting that SREBP is a viable target for pharmacological intervention. The proprotein convertase SREBP site 1 protease (S1P) is an important posttranscriptional regulator of SREBP activation. This report demonstrates that 10 μM PF-429242 (Bioorg Med Chem Lett 17:4411–4414, 2007), a recently described reversible, competitive aminopyrrolidineamide inhibitor of S1P, inhibits endogenous SREBP processing in Chinese hamster ovary cells. The same compound also down-regulates the signal from an SRE-luciferase reporter gene in human embryonic kidney 293 cells and the expression of endogenous SREBP target genes in cultured HepG2 cells. In HepG2 cells, PF-429242 inhibited cholesterol synthesis, with an IC₅₀ of 0.5 μM. In mice treated with PF-429242 for 24 h, the expression of hepatic SREBP target genes was suppressed, and the hepatic rates of cholesterol and fatty acid synthesis were reduced. Taken together, these data establish that small-molecule S1P inhibitors are capable of reducing cholesterol and fatty acid synthesis in vivo and, therefore, represent a potential new class of therapeutic agents for dyslipidemia and for a variety of cardiometabolic risk factors associated with diabetes, obesity, and the metabolic syndrome