Genetic Engineering & Biotechnology News

OCT15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | | OCTOBER 15, 2017 | 25 shakers. This is sufficient material for both preclinical studies and early clinical work. Cell Culture Platform Significant demands are placed on a platform used to develop and scale a cell line from screening in 96-well plates to 5-L flasks. Requirements change as working volumes change as the process moves from screening to development and production. For example, cell culture in 96-well deep- well plates requires far higher agitation and mixing than work in shake flasks or 24-well plates. A system for 96-well plates uses a 3-mm shaking orbit with 1,000-rpm agita- tion, while work in shake flasks and 24-well plates is done using a 25-mm shaking orbit and 100–150 rpm. To compare results from different scales and across multiple instruments, growth conditions must be kept uniform, and pa- rameters influencing cell growth and produc- tivity controlled. 96-Well Plate Cell Culture Considerations Mixing: Standard incubation shakers are not configured for work with 96-well plates. For efficient mass transfer and optimal cell growth in small wells, high-speed mixing and small shaking orbits are required (Fig- ure 3). An earlier study showed that a 3-mm shaking orbit and 1,000-rpm mixing gives higher protein expression and HEK titers of as much as 3,000% higher compared with plates in standard 25-mm orbit shakers and lower speeds. The same study also showed that efficient mixing in plates does not start until 800 rpm. Throughput and Automation: In ATUM's laboratory, the switch to 3-mm shakers and microtiter-plate-based screening allowed a 17-fold increase in throughput and enabled automation of protein purification, simulta- neously freeing up resources and reducing variability due to human error (Table). Evaporation: Evaporative losses must be considered because of the small volumes used in plates and the extended process times (10–14 days). Active humidification is em- ployed to limit such losses over the course of the experiment. Uniformity: During selection of clones grown in triplicates in as many as 80 96-well plates stacked and distributed across a large shaking surface, tightly controlled uniform conditions are of critical importance (Figure 4). The accompanying 3D temperature map of the interior of the Multitron Pro illustrates its ability to keep a consistent temperature throughout. Scaling Up Qualification: The Multitron Pro shaker can be qualified for validated processes. FAT and SAT documentation is available, as is IQ/OQ support. To further mitigate risk to the cell culture, several system modifications are available. Those modifications include antimicrobial coating, UV sterilization of the air flow path, and hygienic steam humidification. Traceability: To allow for interaction with building monitoring systems, the Multi- tron Pro shaker can be equipped with analog outputs for all process parameters. Alterna- tively, the shakers can be controlled using eve ® bioprocess software. This will provide traceability on par with a bioreactor, and al- low both control of complex processes and integration of third-party devices. Also, eve can be qualified for GMP use. Conclusions A machine-learning process, combined with protein engineering and efficient screen- ing facilities, is capable of yielding stable high-expressing cell lines, e.g., a 3–5 g/L IgG1 clone, in approximately 12 weeks. Screening: With up to 7,680 data points (80-microtiter plates) per shaker, it is pos- sible to manipulate multiple factors while performing all experiments in triplicates. This allows for the generation of statisti- cally valid data for design of experiments (DoE), while also considering quality by de- sign (QbD) as stable clones are generated and optimized. Scaling: Stable systems, expressing hun- dreds of grams of protein, can be developed in a matter of weeks. Validation: Systems and process param- eters can be qualified and validated for GLP and cGMP work. Traceability: When the incubation shaker is used in combination with the eve biopro- cess software, traceability is comparable to a bioreactor's. eve also allows full third-party device integration. Bioprocessing Figure 4. This 3D temperature map of the interior of a Multitron Pro shaker illustrates how even the temperature is all across the tray. Figure 3. Mixing in 96-well deep-well plates using a Multitron Pro 3-mm orbit shaker (500 μm/well). Efficient mixing starts at 800 rpm and peaks at 1,000 rpm. Table. Comparison of transient expression of human IgG1 in HEK-293 cells from 1 mL and 10 mL cultures grown under different agitation conditions. Codon optimization was performed using ATUM's GeneGPS. Expression vectors were not optimized using ATUM's VectorGPS. OBTAIN A COHORT UP TO FOUR MONTHS FASTER A novel approach to model generation and breeding: ExpressMODEL™ leverages the benefits of performing IVF on chimeras Produces a cohort in as little as 54 weeks, versus 70 weeks using the traditional approach Bypasses one downstream breeding generation Allows for a more predictable timeline than natural mating Generates a colony at the desired health status Creates the ability to scale up or down deliverables based on project needs TACO N I C .CO M | 1 ( 8 8 8 ) 8 2 2 - 6 6 4 2 L E A R N M O R E A B O U T H OW E x p re ss M O D E L™ C A N R E D U C E YO U R P R OJ E C T T I M E L I N E AT: TACO N I C .CO M / E X P R E S S M O D E L ExpressMODEL ™ PA1086-EN-1709 Tutorial Andrew Magno (a.magno@infors-ht. com) is manager, technical service, Gary Tompkins serves as chief technology officer, and Travis Scagliarini is manager, QA/QC, at Infors USA Inc. Miles Scotcher, Ph.D., is director of business developent at ATUM ( Website:

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