Scale-up: Successful transfer from small to large


Life Technologies PD-Direct® has partnered with Cytovance Biologics to demon­strate rapid process transfer and scale-up using new media developed for fed batch processes and DASGIP Bioreactors. The new media were developed by Life Technologies as cGMP-compliant for a broad variety of cells, and investigations included the optimisation for bioreactor operations and scale-up. A variety of minor adjustments were also made to maximise product yield and cell density and minimise nutrient depletion between feeds.

Life Technologies PD-Direct® developed a fed-batch bioreactor process for a recombinant CHO cell line producing an IgG antibody at 0.6L bioreactor scale. The process description and cell line were transferred to Cytovance Biologics for scale-up to 5L Process Development Bioreactors and to the 100L production bioreactor in the GMP Manufacturing Facility. The feed medium (GIBCO® CHO CD Efficient Feed™ A) was provided as a proto­type of the medium being prepared for commercial launch.

Preparation and procedure

To the extent that scale-up considerations and design differences among the bioreactors would allow, the original bioreactor operating conditions used with Life Technologies’ DASGIP 0.6L bioreactors were used in the Cytovance 5L and 100L bioreactors. No specialised engineering or operating procedures were required to implement the fed batch process.
A volume of “Efficient Feed A” equal to 10% (vol feed/initial culture vol) of the initial culture volume on Day 0 was added on each of the days indicated in the feed schedule for each experiment. No adjustments were made to the feed volume to compensate for changes in culture volume due to sampling and feed additions. Efficient Feed A was added at a rate of 2 mL/minute to the bioreactor to avoid oxygen depletion. Glucose was added to maintain glucose at 2-4 g/L before addition of the feed medium.
The Cytovance contract manufacturing facility uses a 100L stirred tank bioreactor system designed for GMP production. The bioreactor is designed for automated CIP and SIP, and has been fully qualified and validated. The 5L fed batch process was transferred from Cytovance Process Development Laboratories, with parameters scaled as necessary for the production bioreactor. The scaled-up process was incorporated into the Cytovance platform for GMP manufacturing, including development of approved master batch records, and formal quarantine and release of raw materials against approved specifications by Cytovance Quality Assurance.
The Wave Bioreactor cell suspension was used to seed the 100L bioreactor at a working volume of 70L and a cell density of 0.62 x 106 cells/mL. The seeding density was higher than the 5L reference bioreactor run, but was believed to be within the range of acceptable variation for the process. On Days 2, 4, 6, and 8 after inoculation, Efficient Feed A at 10% (v/v) of the initial culture volume was added. The feed medium was added over a period of 3-4 hours to avoid oxygen depletion. The culture was terminated when viability decreased below 50%.

Process transfer and optimisation

At the time the process was transferred, the optimisation of the feed schedule was not complete. The reference 0.6L run in DASGIP bioreactors used a feed schedule of 10% (v/v) CHO CD Efficient Feed™ A feeds on Days 3, 6, and 8. However, glucose was exhausted by Day 5, and glucose, lactate and glutamine were exhausted by Day 9. The Cytovance 5L bioreactor process transfer run used a feed schedule of 10% (v/v) CHO CD Efficient Feed™ A on Days 3, 5, and 7. In addition, glucose supplementation was used as required to prevent the glucose concentration from falling below 1 g/L (Fig. 1). This schedule increased the culture life to 14 days, prevented nutrient exhaustion, and limited waste metabolite accumulation. However, the maximum cell density and product yield were low. The product concentration increased less than 7% after Day 10.

Process scale-up

The feed schedule was changed to 10% (v/v) feeds of CHO CD Efficient Feed™ A on Days 2, 4, 6, and 8 without glucose supplementation for the 5L reference bioreactor run. The new feed schedule achieved the desired result of increasing production and cell density, and of preventing exhaustion, or excessive accumulation, of nutrients and metabolites (Fig. 2). Consequently, the conditions for 5L were used to design operating parameters for the 100L bioreactor run. Although there are differences in the growth profile, by the measures of final product yield, specific productivity, and the general metabolic properties of the cultures, the results in the 5L and 100L bioreactors are very similar. In addition, Life Technologies PD-Direct® obtained very similar results with this feed schedule in their 0.6L DASGIP and 5L bioreactors.

Scale comparisons

A critical issue in process transfer and scale-up is that any significant differences in results among groups and scale of operation can be used to better understand the process. This proved to be true in this project, as shown when Life Technologies PD-Direct™ used the final feed schedule in their 0.6L DASGIP and 5L bioreactors and obtained productivity very similar to the Cytovance bioreactors (Fig. 3).
In spite of cell density distinctions, the impact on nutrient and metabolite concentrations is minimal. The extent to which these differences are a consequence of the high seeding density in the 100L bioreactor is not clear. Although the feed schedule should be linked to the time course of cell density, this process is sufficiently robust that the terminal product yield was stable in spite of the variations between runs.
In both the 5L and the 100L bioreactors, the metabolic properties of the cells made maintenance of a stable culture environment an easily managed process. Control at the acid side of the pH control dead band required only limited additions of base. pH was regulated by the CO2 stripping action of the fixed air sparge. Oxygen demand was satisfied without difficulty. The feed schedule maintained the major nutrients and metabolites in acceptable ranges until exhaustion of the Day 8 feed.
Although there are differences in the cell density profiles between the bioreactors, the final product concentration is remarkably consistent at 900 mg/L and 950 mg/L. Consistent yields were obtained across an approximately 200-fold range of bioreactor volumes (0.6 - 100L) in two unrelated companies, and in three different facilities.D

[1] Source: Jonathan Mitschelen, Christa Short, Pete VanStraten, Jay Peterson, Ricardo

Graniello, John Conner, Steven Perry, and

John Lightholder, 2010,, Successful Transfer and 200-fold Scale-Up of a r-CHO Cell Fed-Batch Process Using a New cGMP Compliant Basal and Feed Medium Combination

Thanks to the co-authors and other people from Life Technologies and Cytovance Biologics for their support.




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