Clark, Colleen E.; (2020) Characterization and Scalability Assessment of a Parallel Single-Use Bioreactor System for Mammalian Cell Culture. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A major challenge in today’s world is the need to discover and develop new medicines with a shortened speed to patient timeline. The pharmaceutical industry is trending towards the use of mammalian cells to produce biologic therapeutics to treat diseases such as cancer. With this comes a need for higher throughput development tools and thus, single-use technologies have found increasing application. Shake flasks and deep well plates are currently used as scale-down models to mimic larger bioreactors. These systems are known to poorly represent large scale bioreactor systems (>1000L), have limitations with oxygen supply, and require considerable manual manipulation. The ability of such small-scale benchtop bioreactors to predict cell growth, productivity, and product quality in larger scale bioreactors is pertinent to meeting speed to patient timelines as well as manufacturing goals. New miniature stirred bioreactor technologies, such as the ambr® 250 disposable bioreactor system, pose a potential solution to the industry’s high throughput need. The ambr® 250 systems enable automated and parallel operation of 24 disposable bioreactors, which can be controlled independently. The amount of user manipulation needed for an ambr® run is significantly less than an equivalent benchtop bioreactor or shake flask experiment. The ability to run these systems with fewer full time employees (FTEs) and produce scalable data makes these systems ideal scale-down tools for high throughput experimentation. The aim of this work was to characterize the engineering environment, cell growth, and antibody production in an ambr® 250 system with a view of defining its role in industrial cell culture process development. Findings were evaluated against current benchtop models (5L) and manufacturing scales (1,000L). Mixing time, power number, and kLa (oxygen mass transfer coefficient) were all assessed to evaluate bioreactor performance and provide quantitative bases for subsequent scale-up studies. These values are known as engineering parameters, which are used to determine the best operating conditions when scaling up and down. When scaling across a large volume range, bioreactor operating conditions must be altered to best fit each system. For example, when scaling up from 5L to 1000L scale, operating conditions can be set by matching parameters such as kLa, mixing time, vvm (volumetric gas flow rate per unit liquid volume per minute), or P/V (power per unit volume). Mixing time was assessed over a range of stirrer speeds from 50-750RPM for both up and down pumping configurations. Mixing times were found to range from ~1 minute at low RPM to <3 seconds at high RPM. A power curve was generated for both up and down pumping impeller configurations using an air bearing technique. The turbulent Power Number for up (1.3) and down (1.4) pumping were experimentally determined for the ambr® 250 bioreactor. This was compared to the vendor published power number of 1.34. kLa values were also determined for a range of stirrer speeds, gassing flow rates, and sparger designs and ranged between 0.9-10 hr-1 for the ranges tested. Characterization comprised the use of computational fluid dynamic (CFD) modeling and validation of simulation predictions using particle imaging velocimetry, power input measurements, and mixing time. A CFD model of the bioreactor was established in M-Star software. Further validation of the CFD model was obtained from experimental measurement of fluid flow throughout the bioreactor using a PIV method. PIV results showed that within the given ranges examined, the bioreactor had no fluid flow dead zones. This is the first time such an extensive validation approach has been used on M-Star software. Cell culture work examined the ambr® 250 as a scale-down model for manufacturing scale tanks. Multiple CHO host cell lines were transfected with mAbs for scale-up/down studies which were based on matching a combination of P/V, vvm, and/or kLa. To achieve this, a rigorous statistical analysis was done comparing multiple scale-down methods including common engineering parameters, altering control strategies, set points, and sparger designs. Multiple transfected mAbs as well as multiple host cell lines were used for this analysis in order to show scalability across a host of mammalian cultures. This work is the most rigorous scale-down work in the ambr® 250 to date. The condition found to best mimic large scale without any geometry change was to operate the ambr® 250 with the open pipe sparger, to stir at 375RPM (down pumping), and utilize a top air flow rate of 0.1mL∙min-1 and a bottom air flow rate of 1 mL∙min-1. Overall, if a geometry change is available, operating with a drilled hole sparger operating at a P/V match of manufacturing scale tanks provided the most scalable process to manufacturing tanks.

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