dramatic improvement in recombinant protein yields using a 96 … · 2018-05-17 · dramatic...
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Dramatic Improvement in Recombinant Protein Yields using a 96-well High Throughput High-Speed Shaking Format and the INFORS HT 3mm Orbit Multitron ProAna Cook, Angela Merluzzo, Cell Signaling Technology, Inc., Danvers, MA 01923
AbstractA growing demand for recombinant proteins has driven the Biotechnology Industry to develop high throughput production and screen-ing methods. Mammalian cell lines are preferable but have been difficult to adapt to 96-well culture due to their high oxygen and CO2 demands. Recently, INFORS HT released the Multitron Pro incubator shaker with a 3mm orbital diameter. By using this instrument at 800–1000 min–1, we have been able to increase recombinant protein yields significantly and improve well-to-well consistency compared to cultures using a 25mm orbit. These gains can be attributed to improved cell growth and viability because of optimal circulation. Ad-ditionally, we investigated different sealing systems and a peg tray that will enable us to stack up to 40 96-deepwell plates per incuba-tor. This will allow for the development of an automatable high-throughput protein expression system that will shave months off of our current development time.
MethodsCell Culture and TransfectionThe 293E cell line, a suspension adapted HEK293 line licensed from the Biotechnology Research Institute, Canada was used in all ex-periments. Cells were cultivated as a seed stock in spinner flasks under the following conditions: 37°C, 5% CO2, and 150 min–1 agita-tion speeds. A 1:1 mixture of F-17 (Life Technologies) and SFM4 Transfix (GE Healthcare Life Sciences) was used as both the seed and production medium. Transfections were performed using a 25-kDa linear PEI (Polysciences) and purified plasmid DNA (Qiagen).
Experimental DesignAll experiments were conducted on 96-deep well deep well plates (Greiner Bio-One Masterblock), and tested two agitation speeds (800 and 1000 min–1) and two working volumes (500 and 1000ul). Transfections were performed in bulk at a 50mL working volume and aliquoted into the final working volume in 96 well deep well blocks. At the indicated time of harvest cultures were removed from the incubators, clarified by centrifugation, and the supernatant samples were analyzed by HPLC for protein expression.
Cell Count and Protein Concentration MeasurementViable cell density and cell viability were measured using a TC 20 Automated Cell Counter (Bio-Rad Laboratories). Cell culture samples from 96-deep well plates were collected on culture days 0–7 for analysis. For protein concentration determination, supernatant sam-ples assayed in replicate using affinity chromatography (Agilent Technologies).
Evaporation Rate DeterminationThe evaporation rate with three sealing systems were compared: Duetz-System (Kuehner Technology), gas permeable membrane sealers (Axygen Scientific), and a Microplate box (INFORS HT). Evaporation was determined by measuring the difference in mass of each plate over 5 days calculated via linear regression and divided by 96 to generate a mg/well*day value. Plates were filled with 1000ul per well and agitated at 1000 min–1 at a 3mm orbital diameter in an 75% humidity incubator.
ConclusionBy using the INFORS HT Multitron Pro incubator shaker with a 3mm orbital diameter, we have been able to increase recombinant protein yields significantly and improve well-to-well consistency compared to cultures using a 25mm orbit. These gains can be attributed to im-proved cell growth and viability because of optimal circulation. Additionally, we tested two agitation speeds, two working volumes, and three plate sealing formats. The optimal parameters were identified to be a fill volume of 1000ul and an agitation rate of 1000 min–1 us-ing a 3mm orbital diameter. By using a membrane-based sealer with the peg tray we were able to maximize throughput in our process. Although other sealing options could have the added benefit of reduced evaporation. Together these findings allow for a high throughput platform for recombinant protein expression.
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Average Protein Yield Improvements Using the INFORS HT Multitron Pro a) Initial experiments utilizing a 25mm shaker inside of an incubator resulted in aver-age recombinant protein yields of 33ug/ml. Using the INFORS HT Multitron Pro incubator shaker, aver-age yields increased dra-matically to 276ug/ml.
a) Cells exhibited typical growth patterns in the INFORS HT Multitron Pro incu-bator shaker comparable to large-scale culture in spinner flasks. Cells grown using a 25mm orbit shaker at 400 min–1 failed to grow appreciably. This effect was most likely due to differences in oxygen transfer2.
b) Cell viability using the INFORS HT Mul-titron Pro incubator shaker was compa-rable to that seen in large-scale spinner flasks. In contrast, cell viability using a 25mm orbit shaker at 400 min–1 dropped appreciably one day post seeding and continued to decrease for the duration of the experiment.
A comparison of three commercially avail-able plate-sealing formats resulted in sig-nificant differences in the residual fluid volume following five days of culture in the INFORS HT Multitron Pro. The least evaporative loss was seen using the Du-etz-System followed by the Microplate Box (INFORS HT). Membrane based seal-ers resulted in the highest level of evapo-rative loss which was roughly 30–40% depending on the various culture formats.
b) Further analysis of different high and low yielding targets confirmed that the effect was consistent for multiple proteins.
a) No significant differences in protein yields were observed using the three sealing methods.
b) Over all conditions, the protein con-centrations were higher in wells filled with 500ul of total culture compared to those filled with 1000ul (Data not shown). However, when taking into ac-count the relative differences in fluid volume, the total yields observed in the 1000ul culture were significantly higher.
c) Across all conditions, increasing agitation speed from 800 min–1 to 1000 min–1 in-creased protein yields.
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Protein Yield Improvements Across High and Low Yielding Targets
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Figure 3: Differences in protein yield resulting from sealing method, fill volume and agitation speed
Figure 4: Evaporative loss using different sealing formats
Figure 2: Increased oxygen transfer enhances cell growth
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Differences in Protein Yield Resulting From Sealing Method, Fill Volumeand Agitation Speed
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For Research Use Only. Not For Use in Diagnostic Procedures. © Cell Signaling Technology, Inc. CST and Cell Signaling Technology, are registered trademarks of Cell Signaling Technology, Inc. All other trademarks are the property of their respective owners.
Figure 1: Recombinant protein yield improvements using the INFORS HT Multitron Pro incubator shaker with 3mm orbital compared to a 25mm orbital shaker.
Reference1 www.thermoshaker.com/?tag=orbital-shaker2 Bos, A.B., Luan, P., Duque, J.N., Reilly, D., Harms, P.D., Wong, A.W. (2015) Biotechnology and Engineering. 112 (9)
1832-1842.
Ana Cook, Cell Signaling [email protected]
Angela Merluzzo, Cell Signaling [email protected]
www.cellsignal.com
Infors AG,Headoffice, SwitzerlandRittergasse 27, CH-4103 BottmingenT +41 (0)61 425 77 00, F +41 (0)61 425 77 [email protected]
www.infors-ht.com
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