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Thomson Instrument Company is not affiliated with GSK, Genentech (a fully owned Roche company), Pfizer Inc, Biosilta's Enbase media, Life Technologies and its Magic Media , Kuhner AG, and New Brunswick Scientific.
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Abstract
The patented Ultra Yield Flask™ is a novel, disposable shake flask design that supports high-density culture growth in rich media. The Ultra Yield Flask achieves this by using a novel near-vertical wall angle coupled with a six baffle design that has been optimized for the fermentation of E. coli, P. pastoris, S. pombe, Schizomycetes, and other microbes. Under suitable conditions, the Ultra Yield Flask™ generates up to ten times greater oxygenation compared to traditional shake flask designs. A companion AirOTop air-porous seal is used to cover the flask from potential culture contamination, rather than traditional cotton plugs or aluminum foil. The Ultra Yield Flask provides scalable culture growth results when using the 125mL, 250mL, 500mL, or 2.5L design.
Introduction
How does the yield increase with Ultra Yield Flasks?
The Ultra Yield Flask™ improves yield. Because the Ultra Yield Flask™ allows increased cell densities to be achieved over traditional shake flask designs, this results in increased yields of biomass and concomitant improvement in yields of recombinant protein or DNA. Cells in Ultra Yield Flasks™ may be grown for more than 24 hours if necessary, as the cultures are not oxygen limited. Cells do not go directly from log phase to stationary phase but pass through a transition phase, as shown by data from GSK (see GSK data). This transition stage allows for continued growth without anerobic conditions, and uses the cellular nutrients to maintain viability. pH maintenance is extremely important during this phase, and buffering with a non-temperature-dependant buffer, e.g. MOPS, may make a large difference in cell growth behavior when using the Ultra Yield Flask™.
The high growth observed with the Ultra Yield Flask™ has been found to be scalable across the different size variations, from the 125mL flask to the 2.5L flask. Studies have shown that the Ultra Yield Flask™ provides consistent batch to batch results.
Ultra Yield Flask™ volumes and recommended orbital speeds for culturing E.coli and other microbes. The culture volumes deployed in Ultra Yield Flasks™ is somewhat dependent on cell type for optimal performance. A general rule for E.coli is to use a 40% fill volume. End-users have deployed 500mL for maximum OD, or 1L for additional production in the same footprint. Often, 3-4 flasks may be used to replace a traditional small scale fermentor.
Recommended speeds:
Recommended culture volumes and orbital shaker speeds (a throw of 1" is generally used for orbital shaking)
| Part # | Description | Media (mL)/Flask |
Shaker Speed |
|---|---|---|---|
| 931147 Please cover the flask with AirOtop seals 899415. |
125mL Ultra Yield Flasks 50/case -- Sterile |
40-50mL/flask | 300-350 RPM |
| 931144 Please cover the flask with AirOtop seals 899415. |
250mL Ultra Yield Flasks 50/case -- Sterile |
75-110mL/flask | 300-350 RPM |
| 931141 Please cover the flask with AirOtop seals 899415. |
500mL Ultra Yield Flasks 50/case -- Sterile |
125-200mL/flask | 300-350 RPM |
| 931136-B Please cover the flask with AirOtop seals 899414. |
6/case 2.5L Ultra Yield Flasks -- Sterile |
500mL (optimum) |
300-400 RPM |
| 931136-B Please cover the flask with AirOtop seals 899414. |
6/case 2.5L (Fermentor Mode)-- Sterile | 1L | 250 RPM |
Results
Genentech, Inc
GSK
Pfizer
610% Yield Increase*
Effect of flask design on E. coli culture growth and production of recombinant protein. (A) The effect of flask type and growth medium on the observed optical densities of the cultures at A600 following overnight protein expression. Cultures carried out in Fernbach flasks using either LB medium or TB medium are shown, respectively, at the front (green) and in the center (orange). Cultures grown in Ultra YieldTM flasks in TB medium are shown at the back (blue). See Table 1 for a list of the recombinant proteins overexpressed by the various clones. (B) The yields of expressed soluble protein (determined by protein assay and recorded as mg protein per liter of expression culture) from the IMAC columns following purification of the twelve polyHis-tagged recombinant proteins, each expressed under the three conditions described in Panel A. Clone locations in Panel B are the same as those in Panel A
*Economical parallel protein expression screening and scale-up in Escherichia coli. Journal of Structural and Functional Genomics2006 Jun;7(2):101-8. Epub 2006 Dec 23.
Conclusion
The Ultra Yield Flask™ offers a simple alternative to traditional shake flasks, and to complex small-scale (‹10L) fermentation devices at minimal cost. The Thomson Instrument Company, together with users at Genentech, GSK, and Pfizer, have validated the Ultra Yield Flask™ as a suitable system to achieve high-density cell fermentation. Pfizer scientists have demonstrated that the Ultra Yield Flask™, on average, allows greater cell densities to be achieved in a similar footprint by up to 350%, and that this was mirrored by a similar return, on average, of 610% in recombinant protein yields (Brodsky, O. & Cronin, C.N. [2006] J. Struct. Funct. Genomics. 7, 101-8). These data demonstrate that the Ultra Yield Flask™ substantially reduces the protein production footprint, and may make traditional fermentation approaches unecessary at smaller scales.
Fully automated fermentor systems available in the market today range from $15,000 to $90,000 dollars. However, the simple and inexpensive Ultra Yield Flask™ solution can slash that price to only a few dollars which fits in any large or small research budget. Apart from the obvious fiscal advantage, other advantages include the ease of use (no requirement for detailed fermentation set-up and sterilization runs), the fitting of the flasks in standard laboratory floor shaker systems, and the simplicity of construction. It is anticipated that the Ultra Yield Flask™ will become the flask of choice for recombinant protein production in the research setting.
Materials and Methods
What media do we put in the Ultra Yield Flasks?
An enriched media with a pH balance around 7.4 is recommended. Terrific Broth (TB), EnBase, and auto-induction media, such as Magic Media (InVitrogen), are suitable examples of enriched media. The pH balance is most useful with buffers that contain phosphate since these are not temperature dependant. MOPS at 100 mM is a suitable alternative to phosphate-based buffers.
Is the Ultra Yield Flask™ disposable or re-usable?
The Ultra Yield Flask™ is priced to be a disposable bioreactor or disposable shake flask. The Ultra Yield Flask™ may be reused following sterilization. However, the cost of the flask is significantly cheaper than the effort required for autoclaving (with labor at ~ $100.00/h it does not make sense to reuse the product?). Nevertheless, if one wishes to reuse the flask it is possible to do so a number of times. However, multiple autoclaving runs will make the flask more brittle and likely to fail. A limit of three uses is suggested.
Myths and legends of growing cultures?
- LB is the least healthy growth media. High density microbial growth will only be achieved in the Ultra Yield Flask™ when using an enriched medium, such as Terrific Broth (TB).
- Growing cultures for more than 18h will damage the culture. Cell damage and inferior growth is generally the result of anaerobic conditions resulting from a lack of oxygen, and a drop in pH when using traditional shake flask designs. The Ultra Yield Flask provides sufficient aeration to maintain cell growth for up to 7 days at 20°C, and up to 50h at 37°C.
Why are LB and Minimal Media unsuitable for the Ultra Yield Flask™?
LB or Minimal Media do not contain sufficient nutrients to support microbial growth to high cell densities. The Ultra Yield Flask cannot differentiate itself from Traditional shake flask designs when using low growth media, and all flasks will perform in a similar fashion. However, traditional shake flask designs cannot approach the cell densities that are achievable in the Ultra Yield Flask™.
What orbital shaker equipment are suitable for use with the Ultra Yield Flask™?
Most shaker platforms support high density cell growth in the Ultra Yield Flask, including those from New Brunswick (the Model 44R is not recommended), ATR and Kuhner.
