Cell culture media mixing in a benchtop LevMixer™ single-use mixer

For each experiment, we installed and set up the LevMixer™ 10 L single-use mixing system according to the instructions for use.

We then placed the single-use biocontainer bag in the mixer and deployed to obtain a final 3D and cubical shape after which we calibrated the single-use pH sensor with the precalibrated label.

The single-use biocontainer bag was filled with 9 kg of demineralized water (considering a density of 1000 kg/m³) to reach a volume of 90%. Then, mixing was started at the max speed of 2900 rpm, at room temperature (20°C ± 2°C) and without control. After 1 min of stable mixing, we added the media through the powder port located on top of the mixing system.

Individual procedures provided with the media were then followed. The recommended dissolution time for the HyClone™ peak expression medium was of 20 min and 10 min for the HyClone™ DMEM high-glucose concentrations. Once the powder was dissolved, we supplemented media with the right amount of sodium bicarbonate to achieve a certain concentration and pH (Table 1).

As a next step, around 10 min after, the speed was reduced to 2000 rpm and sodium hydroxide (for the HyClone™ peak expression medium) or hydrochloric acid (for HyClone™ DMEM high-glucose medium) were added to adjust the pH to the targeted range of 7.0 to 7.40.

After a stabilization time of around 10 min, the remaining volume of water was added to obtain the final solution of 10 L at the right concentration.

We filtered the resulting solution over a sterilizing grade Supor™ Prime filter using a peristaltic pump. Pressure was monitored to evaluate the possible fouling of the membrane.

We used the Supor™ EX ECV sterilizing grade filter in Mini Kleenpak™ capsule as a reference filter. This is a typical polyethersulfone (PES) media filter.

The filter capacity was determined by P_max analysis, see below.

We tested filter integrity post-filtration using a bubble-point (BP) test, see below.

Table 1. HyClone™ media components and concentrations

Mixing times

We evaluated mixing times from the time when the powder mix was added until it was visually dissolved (visual mixing time) and with the time taken to achieve 95% mixing (T₉₅) when the pH values have stabilized within the range of ± 5% of the end value (95%; 105%).

Fig 2. Example of a chart showing a pH shift with the T₉₅ and stable reading in the range of ± 5%.

Sterile filtration P_max

Our filterability test was performed at a constant flow rate using a peristaltic pump in the test setup shown in Figure 3.

Fig 3. The P_max test setup.

The results obtained were extrapolated using direct linear scale-up calculations based on the ratio of filter area between the test system and the process-scale filter. This is applicable when the provided process fluid sample volume allows for a filtration volume through the test filter system directly equivalent to the process-scale volume. An extrapolation method, P_max, may be applied to trials where equivalent sample volume is not filtered, either due to sample volume or time constraints.

P_max calculations cannot be applied when no increase occurred and/or the course of pressure increase cannot be fitted to the underlaying model (resistance model).

Method procedure constant flow

We installed a 3 cm² Supor™ Prime sterilizing grade filter (DC3) or a 2.8 cm² Supor™ EX ECV sterilizing grade filter (KS2) in a filter capsule and connected them to a product reservoir containing the fluid sample. We filled the reservoir with the sample, and set the peristaltic pump to deliver flow rates of 7 and 14 mL/min. Once the system was ready, the pump was activated and the manual valve opened to initiate the flow. A beaker was placed on a tared balance to collect the filtrate. The flow rate was then adjusted to a scaled-down value based on customer requirements and equipment limitations. The pump was started again, and the timer initiated simultaneously.

Throughout the filtration process, key parameters such as upstream pressure, filtrate volume, filtration time, conductivity, and turbidity were monitored and recorded at fixed intervals determined by the product's filterability. The test concluded when one of the predefined stop criteria was met.

Sterile filtration integrity test: Bubble point (BP)

We assessed the integrity of the test filters with the BP nondestructive integrity test method and the filters wetted with water. The reference value for the BP was 4.1 bar (60.2 psi, 0.4 MPa). This value corresponds to the lowest BP value of a test filter that fully retained Brevidimonas Diminuta (B. Diminuta) during bacteria retention testing.

The BP test is designed to detect the largest pores of a membrane filter. It is based on measuring the gas flow across a completely wetted membrane at increasing gas test pressure, until the point at which the wetting fluid is expelled from the pores, and bulk flow is measured.

In Figures 4 and 5, you can see the evolution of the pH reading per step in the mixing process for each medium.

Fig 4. Chart of the pH reading recorded for the HyClone™ peak expression medium preparation in the LevMixer™ 10 L single-use mixing system.

Fig 5. Chart of the pH reading recorded for the HyClone™ DMEM high glucose preparation (4500 mg/L) in the LevMixer™ 10 L single-use mixing system.

Table 2. Process information and mixing times

Table 3 and Table 4 show the throughput at a constant flow rate of 7 and 14 mL/min for both the Supor™ Prime filter and a benchmark Supor™ EX EKV polyether sulfone (PES) filter.

The pressure increase noted between the different flow rates was only due to the increase in flow rate and not a higher fouling occurring as the pressures stayed stable throughout the filtration.

Table 3. Throughput, sterile filtration of HyClone™ expression medium

^* Filtered at a constant flow rate of 7 mL/min

^† Filtered at a constant flow rate of 14 mL/min

^‡ 1 bar = 14.5 psi = 1 MPa

Table 4. Throughput, sterile filtration of HyClone™ DMEM high-glucose medium

^* Filtered at a constant flow rate of 7 mL/min

^† Filtered at a constant flow rate of 14 mL/min

^‡ 1 bar = 14.5 psi = 1 MPa

Table 5 shows the integrity test results. We assessed the integrity of the test filters with the BP nondestructive integrity test method and the filters wetted with water. All filters used in this study passed the integrity test.

Table 5. Integrity test results

^* 1 bar = 14.5 psi = 1 MPa

^† Filtered at a constant flow rate of 7 mL/min

^‡Filtered at a constant flow rate of 14 mL/min

We measured conductivity of the media to verify if the filtrations influenced the initial properties of the media (Table 6).

Table 6. Conductivity, pH, and temperature of the media tested at different flow rates on benchmark filter and post Supor™ Prime sterilizing-grade filters used at different flow rates

The contributions made to this application note were made by Mustapha Hohoud, Senior Engineer Bioprocess, and Stijn De Backer, Downstream Field Application Specialist.