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FIFTYOD - Fermenter in a Flask Growth Medium for E. coli

Production_environment_prozomix.gifNEWFIFTYOD - very high cell density growth medium for E. coliHigh density E. coli BL21 growth medium - mediaDownload .pdf

Recombinant protein production facility at Prozomix (capacity 360 x 2 L flasks).  Central shaker has inocula (bottom shelf) for new FIFTYOD production run (flasks being loaded onto top shelf of shaker furthest in view).The front top shelf of central shaker is a production run nearing harvest point, comprising 20 x 0.65 L FIFTYOD cultures at an optical density of approx. 58 at 600 nm.  After processing, 130 g of freeze-dried cell-free extract powder was produced from this particular production run.  Prozomix has been using FIFTYOD to effect its recombinant protein CMO business for more than 2 years, during which time the composition of the medium has been improved significantly, and relaible protocols established.

The Challenge: producing small quantities of recombinant protein is these days a relatively straightforward process, given a plethora of expression systems, synthetic DNA technologies, and widespread abundance of general laboratory equipment and expertise required.  A number of commercial services, such as those provided by Prozomix, also offer cost-effective solutions in this area.  However, when a requirement for recombinant protein exceeds around 1 g and heads for 100 g and beyond, typical academic laboratories are frequently out of their comfort zone.  At this stage the bottleneck is generally culturing a sufficient volume of E. coli to produce the required quantity of recombinant protein.  Even where fermenters are accessible, via for example a central academic service, such facilities are frequently expensive to access with no guarantee of success, quality or timely delivery, as such facilities are intended for academic, rather than commercial performance (as a rule).

The Solution - "Fermenter in a Flask": fermenters are an excellent solution for producing larger quantities of recombinant protein using expression hosts such as E. coli BL21, as they can add alkali or acid to maintain optimal pH, apply an optimal temperature profile, feed growth components and supply oxygen (typically in the form of air) efficiently to the growing/expressing cells.  With this control and in conjunction with rich or fed growth media (fedbatch fermentation), cell densities some 20-30 times greater than reached in typical LB growth medium are frequently achieved (i.e. an optical density in the range of 50-100 at 600 nm), meaning that a 20 L fermenter, operating at 15 L working volume, typically produces approx. 150 g of crude recombinant protein preparation (freeze-dried cell-free extract powder basis).  However, if a fermenter can not be employed, producing the same 150 g of crude recombinant powder would become a real task, as it would take the equivalent of at least 300 x 0.5 L culture volumes of LB to achieve the same amount of expressing bacterial cells!  In an attempt to somewhat address this issue, higher density growth media have been developed and are available from a number of suppliers, yet in practice the achieved cell densities for some of these products is only in the region of A600 10-15 at best.  A further development in the right direction was the development and commercial offering of true high cell density growth media, specifically in the A600 50-70 range.  The commercial providers of these products offer a good solution for many laboratories with a requirement for scaled production of recombinant proteins from E. colibut some such commercial products are still somewhat limiting as:

(1) they can require high rpm shakers

NOTE: just 140 rpm required for FIFTYOD growth medium from Prozomix 

(2)  only small culture volumes can be grown (e.g. 0.1 L per plask)

NOTE: 0.75 L per 2 L flask can be used for FIFTYOD growth medium from Prozomix

(3) they can require specialist flasks

NOTE: common baffled 2 L flasks can be used with FIFTYOD growth medium from Prozomix

(4) they can require a license

NOTE: no license is required for use of FIFTYOD growth medium from Prozomix

(5) they can require growth component additions at certain growth stages

NOTE: no such additions required for FIFTYOD growth medium from Prozomix

(6) they are not animal free / BSE-TSE Certifiable

NOTE: FIFTYOD growth medium from Prozomix is animal free / BSE-TSE Certified

(7) they are not associated with a scaled-out contract manufacturing service employing the same growth medium

NOTE: Prozomix offers an up to 1000 L scaled-out FIFTYOD growth medium based CMO service to produce up to 10 kg of "same as" recombinant protein preparations at known list prices

 

Please contact sales@prozomix.com for further information.