micro-MatrixMonitored CHO cell batch in 24 parallel micro-bioreactors

a step ahead

IntroductionCurrently, the implementation of scale-down methods for rapid development and optimization plays an important role in the research of new therapies and the cost-effective drug development process. (Barrett et al., 2010). The 24 standard round well (24 SRW) has already been established as a suitable format for the cultivation of mammalian cell lines and successfully used as a scale-down model for shake flasks (Micheletti & Lye, 2006; Silk et al., 2010). Although the traditional microtiter plates offer a high degree of parallelisation at an affordable price, they lack the monitoring and control of pivotal process parameters like pH, dissolved oxygen (dO2), and temperature (T), leaving the operator with a black-box instrument. Applikon’s micro-Matrix bridges this gap by providing full monitoring and control of these culture parameters. The platform holds 24 individual micro-bioreactors based on a 24 deep-well microtiter format. In this study, Applikon’s micro-Matrix was used to monitor the batch culture of the GS-CHO cell line CY01 under pH (down-control), dO2 (up- and down-control) and T controlled conditions. The results obtained reveal reproducible behaviour in the 24 wells in terms of parameter profile and show that additional up-control for pH is needed during the process. Offline measurements were also done such as viability, growth kinetics, glucose consumption and titer production. Additionally, evaporation resulted lower in the micro-Matrix than in the microtiter plate (results not shown) and suggests that dO2 control loop could be beneficial for cell culture. In this study, a CHO cell batch was monitored in order to gain a better understanding of the process to be used in other applications, such as fed-batch culture. The micro-Matrix proved to be a suitable tool to achieve a fully-monitored and controlled environment for micro-scale cell culture applications.

Materials and methodsPrecultureCells were thawed and expanded in a 250 mL shake flask with a vent cap (Corning Life Sciences, USA) for 7 days in CD-CHO (Life-Technologies, UK) with 25 µM MSX (Sigma-Aldrich). The shake flask was mounted on an orbital shaker (Sartorius, UK) with an

orbital diameter of 25 mm and a shaking speed set to 150 rpm and incubated at 37°C, 5% CO2, and 70% humidity.

Batch culture in the micro-MatrixA suspension with a final concentration of 0.3·106 viable cells/mL was prepared using the appropriate amount of CD-CHO medium. The volume of 3.5 mL of this suspension was used for the 24 deep square well cassette (Applikon, The Netherlands). The micro-Matrix cassette was covered with the top plate, connected to the gas supply lines and clamped onto the Optical thermal module (OTM) of the micro-Matrix (Figure 1). The set points were specified at pH 7.2, 30% dO2, and 37°C. Down-control of pH was achieved with the addition of CO2, the dO2 was two-sided controlled through the addition of compressed air and N2. The shaking speed was 220 rpm.

Analytical methodsViable cell concentration and viability were determined using the Vi-CELL XR (Beckman Coulter, UK), which is based on the Trypan Blue staining method. Samples were diluted 1:1 with Phosphate Buffered Saline (PBS) (Thermo Fisher Scientific, UK)). Metabolite concentrations were determined using the NOVA Bioprofile Flex (nova biomedical, UK). Samples were diluted 1:1 with Milli-Q water. IgG4 quantification was done using an Agilent 1200 (Agilent Technologies, UK) high-performance liquid chromatography (HPLC) with a 1 mL HiTrap Protein G HP column (GE Healthcare, UK). A buffer of 10 mM NaH2PO 4 (Fluka, Cat No. 71642-1KG) and 10 mM NaH2PO 4·H2O (Sigma-Aldrich, Cat No. 71507-1KG) adjusted to pH 7 and a buffer of 20 mM glycine (VWR, Cat No. 101196X) adjusted to pH 2.8 was used to operate the column. The elution peak was recorded via UV at 260 nm and a standard was used to calculate the resulting IgG4 concentration. Where applicable,the analytical results were corrected for evaporation.

Figure 1A | Representative well of a micro-Matrix cassette, covered with the top plate, fitted filter bars, and a microvalve of the Liquid Delivery Module for automated liquid feeding. Optical sensors for dO2 and pH are located at the bottom of the well. Temperature sensor as well as heater and cooler are part of the OTM. Figure 1B | The microtiter system comprises of an inbuilt orbital shaker with a shaking diameter of 2.5 cm, which is placed in a temperature controlled chamber. The micro-Matrix cassette sits on top of the OTM and is connected to pressurised feed bottles and a gas supply.

Results and discussionParameter controlThe micro-Matrix could perform well monitoring and control of pH, dO2 and T and the reproducibility of results in the 24 wells is shown in Figure 2. The pH dropped during the process to 6.5, showing that up-control of pH is needed to maintain the set point at 7.2.

References• Barrett TA, Wu A, Zhang H, Levy MS, Lye GJ. 2010. Microwell engineering characterization for mammalian cell culture process development. Biotechnol. Bioeng. 105:260–275.• Micheletti M, Lye GJ. 2006. Microscale bioprocess optimisation. Curr. Opin. Biotechnol. 17:611–618.• Silk NJ, Denby S, Lewis G, Kuiper M, Hatton D, Field R, Baganz F, Lye GJ. 2010. Fed-batch operation of an industrial cell culture process in shaken microwells.

Biotechnol. Lett. 32:73–78.

1A 1B

Growth kineticsCell concentration steadily increases during the first 6 days (Figure 3A). Subsequently, the viable cell concentration remains stagnant for 2 days. The maximum cell density is reached on day 10 with 4·106 cells/mL. The viability stays above 90% for the initial culture period and starts to decline after about 8 days of cultivation to approximately 53% on day 13 (Figure 3B).

Figure 3A | Growth kinetics ( ) and 3B viability ( ) for CHO cells grown in the micro-Matrix. Error bars represent one standard deviation (n = 3).

Production kineticsFollowing the lag phase in the first 4 days of cultivation, the titer rapidly increases until day 10 and reaches its maximum value of around 0.6 g/L. on day 14 (Figure 4). The highest productivity coincides with the end of the logarithmic growth phase. Productivity of this cell line can, therefore, be considered non-growth associated.

Metabolic profileThe concentration of glucose steadily decreases from 5.7 g/L and reaches depletion on the 10th day of the cultivation (Figure 5A).The concentration of lactate drastically increases within the first 4 days of the cultivation and then plateaus for 2 days (Figure 5B).Subsequently, lactate is utilised by the cells and its concentration decreases as a result before it reaching depletion on day 10.

On day 11 and 13, a slight increase of the lactate concentration can be observed.

Figure 5A | Glucose consumption ( ) and 5B Lactate production / consumption ( ) for CHO cells grown in the micro-Matrix. Error bars represent one standard deviation (n = 3).

EvaporationEvaporation steadily increases and reaches a maximum of 30% on day 14 of the cultivation (Figure 6). As can be seen by the error bars, some wells are subject to substantial variability in the evaporation. Unlike conventional microtiter plates, this variability

appears to be independent of the well position.

Conclusions• The micro-Matrix was successfully used to cultivate the GS-CHO cell line CY01 with the advantages of the monitoring and

controlling of culture conditions.• The pH, dissolved oxygen and temperature profiles were reproducible in the 24 wells. • pH was well controlled but up-control is also needed. • Productivity is non-growth associated and coincides with stagnant viable cell concentration and onset of lactate consumption. • The lactate profile follows the typical pattern of production / consumption for CHO cells.• Evaporation is lower than conventional microtiter plates meaning that dO2 control loop is beneficial for cell culture.

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Figure 4 | Production kinetics for CHO cells grown in the micro-Matrix ( ). Error bars represent one standard deviation (n = 3).

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Figure 2 | Parameter control in micro-Matrix for the 24 wells, A) dissolved oxygen profile, B) pH profile and C) temperature.

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Figure 6 | Percent of well content evaporated for CHO cells grown in the micro-Matrix ( ). Error bars represent one standard deviation (n = 3).

6

Wiegmann, V1., PhD-researcher, Bernal, C2., PhD, Kreukniet, M2., Msc, Baganz, F1., Prof. 1 Department of Biochemical Engineering, University College London, Gordon Street, WC1H 0AH, London, U.K.

2 Applikon-Biotechnology BV. Heertjeslaan 2, 2629JG. Delft, The Netherlands.

micro-Matrix 3.5 mL fill volume

micro-Matrix 3.5 mL fill volume