1 Introduction
Traditionally, bioreactor performance is quantified by considering the critical transport processes and the biochemical conversion process itself [1]. Since most industrial bioprocesses are aerobic, oxygen is a limiting nutrient for cell growth and metabolic production; thus, oxygen transport into the liquid phase is a limiting factor in bioreactor and overall process performance [2]. It is therefore critical to maintain adequate transport of oxygen from the gas phase into the culture medium. Commonly, characterization of bioreactor oxygen transport is presented as the volumetric mass transfer coefficient, kLa which can be used to compare oxygen transfer performance from the microscale to industrial volumes [2,3] and is an established criterion for predictive process scale-up [4,5].
With the recent development of cell-based therapies to treat a wide range of unmet clinical needs, pharmaceutical companies have significant pressure to introduce new products to the market as quickly as possible [6]. When developing new therapies, early stage process development and optimization requires extensive screening of culture conditions to maximize cell growth and viability, with thousands of experiments optimized across hundreds of variables and the best performing conditions transferred to downstream production. This screening process is time consuming and expensive, due to the high cost of media components and often reduced cell growth of the cellular therapy cell types [7]. As a result, it is advantageous to implement a scaled development process wherein testing of the broadest parameter space takes place in the smallest possible volumes with promising candidates then transferred to larger volumes for additional testing.
Microplates present an ideal format for early process development due to their low working volumes (µL-nL) and throughput and automation capabilities. However, the utility of microplates for high throughput process development is limited due to poor control of oxygen transfer or kLa (h-1) in these formats. Ideally, it would be possible to control the kLa in microplates to match those seen in larger volume mammalian cell culture systems, such as the Sartorius ambr® 15/250 (15-250mL) and Cytiva’s Wave Cellbag (2L-200L). For the former, kLa value ranges are of 0.18 - 7.90 h-1 and 2.15 -11.52 h-1 are predicted while the latter can achieve kLa values that range from 1-60+ h-1 [8,9].
Mechanical shaking is the only commercially deployed solution for microplate mixing of cultures, and its utility is limited. Microplate shaking [10] induces splashing motion by overcoming surface tension. Splashing is an inertial effect, and as microplate well size decreases, the inertia of the droplet decreases, while surface tension remains the same. Thus, smaller wells require greater shaking energy (amplitude, frequency) to achieve the same mixing performance. For 96 well plates this critical threshold has been observed to be approximately 800 RPM (for a 3mm shake amplitude) [10,11]. Achieving the desired results from mechanical shaking is surprisingly challenging. While kLa in a conventional bioreactor improves smoothly with increasing impellor speed [12], shaking performance depends on whether the shaking is in-phase or out-of-phase. Increased frequency has little impact on out-of-phase mixing, and the harmonic frequency of microwells varies dramatically with well size and shape [11]. Due to these limitations, as well as concerns related to shear stress induced damage at high shake frequencies, commercially available incubator shakers are not marketed for use with mammalian cell culture. Shake-less methods such as acoustic mixing exists, but adoption has been limited by concerns about heating and scalability. In short, gentle microplate agitation remains a major unmet need for bioprocess discovery and development, especially for mammalian cell culture.
This research reports the development and characterization of a novel microliter scale culture plate with novel surface-attached agitation methodology. The microplate is designed for high throughput process development applications due to its operation in a 96-well culture plate with culture volume of 150-300 µL allowing for adequate sampling for bioanalysis. Evaporation of culture media during operation, which has been shown to be an issue in small volume culture reactors [13], is compensated for by operation in a high humidity environment. The oxygen transfer performance of the microreactor is benchmarked against conventional small-scale culture systems.