In process development, appropriate scaling is important to achieve acceptable product quality without compromising titer (1). Scale-down approaches involve matching the oxygen transfer coefficient (k_La) value, impeller tip speed, power per unit volume, or mixing time to those of a bioreactor (2). Bench-top bioreactors are typically used in bioprocess engineering as scale-down models of commercial units in fermentation and cell culture because of their similarity in geometry (H/D ratio) and mechanical properties (agitation type and sparging). By contrast, shaking culture systems such as Wave bioreactor (GE Healthcare), shake flasks, and tube spins are typically used for culture expansion.

Significant improvements to the composition, shape, size, and caps of shake flasks have facilitated sufficient oxygen transfer and mixing (3). Such modifications have allowed for use of shake flasks in production cultures for clone evaluation, media development, and process optimization. But discrepancies in cell culture performance with respect to growth, lactate, and productivity (which could be attributed to differences in pH and dissolved oxygen, DO) are frequently observed between shake flask and bioreactor cultures (4,5). So further improvements are needed before shake flasks can be more reliably used scale-down models for process development.

PRODUCT FOCUS: PROTEINS, ANTIBODIES

PROCESS FOCUS: UPSTREAM, PRODUCTION

WHO SHOULD READ: PROCESS ENGINEERS, QA/QC

KEYWORDS: PAT, PROCESS OPTIMIZATION, SCALE-DOWN, K_LA, fed-batch culture, pH, dissolved oxygen

LEVEL: INTERMEDIATE

Although pH in shake flask cultures can be measured offline, it is not convenient to monitor it routinely. Sensor technology has evolved from fiber-optic probes (which are fragile when mechanically stressed and require autoclaving) to noninvasive sensors that can be affixed to sterile vessels (6). Advances in noninvasive fluorescence sensor technology have enabled the ability to monitor pH and DO values at a small scale. Methods for determining pH and DO values using noninvasive sensors include frequency-modulated excitation, fluorescence lifetime, dual-lifetime reference, and ratiometric measurement (7,8).

Sensor patches have evolved and are now <1 mm in size. Costs of sensors and patches have dramatically decreased, and they are widely used on a range of vessel types (9). Several commercially available systems use noninvasive sensors complemented with online monitoring capability for different types of cultures. Models include the PreSens Shake Flask Reader (SFR) system (Precision Sensing GmbH) and SENSOLUX stand-alone version (Sartorius Stedim Biotech) for shake flasks and Micro-24 miniature bioreactor system (Pall) and ambr miniature bioreactors (TAP Biosystems). In addition, the same technology has been used at larger scale in the Wave bioreactor and the Single Use Bioreactor (SUB) (HyClone) system.

Here, we compare performance of cell cultures run in shake flasks and bioreactors and present the necessity for pH and DO monitoring and control. We selected the PreSens SFR shake flask online monitoring system to investigate culture pH and DO differences among shake flasks and bioreactors.

Table 1 compares capabilities and costs of the PreSens system with those of a typical shake flask system and a 2-L bioreactor. Although it is more costly, online monitoring functionality warrants the additional cost. We tested the stability and accuracy of the pH and DO sensors to determine instrument functionality and calculated k_La values for shake flasks under different operating conditions.

Table 1: Comparing a traditional shake flask system, a PreSens shake flask system, and a 2-L bioreactor system (Click to enlarge)

We proposed operating conditions to match k_La values at large scale. We monitored the pH profile after bolus base addition (the traditional method of pH control in shake flasks) to determine the extent of the addition's influence on culture pH. We examined the effect of a lack of agitation during shake-flask sampling on pH and DO profiles. Results elucidate the potential differences in shake flask cultures compared with bioreactor cultures.

Materials and Methods

The PreSens SFR system is a noninvasive online system for monitoring pH and DO for as many as nine shake flasks. The presterilized, precalibrated polycarbonate baffled shake flasks with vented caps have two immobilized sensor spots — one for pH and one for DO — affixed to their interior flask bottoms. The shake flasks are clamped onto the SFR, which houses an integrated optical module per shake flask for wireless data transmission to a PC for collection and visualization (Figure 1). The DO sensor uses a phase-modulation technique that correlates the luminescent decay time with a quantitative value of DO, calculated with the Stern-Volmer equation (8). The pH sensor uses an internal-referenced measurement with a patented combination of fluorescent dyes to detect changes in intensity and correlate it with a pH value. Given ranges for sensor detection are 0–100% air saturation for DO and 5.5–8.5 for pH.

Equation 1:

Figure 1: (Click to enlarge)

Stability and Accuracy: We tested the stability of the pH sensor by monitoring the change in pH value of phosphate buffered saline (PBS) in a 250-mL shake flask over 12 days with a 15-second interval online sampling frequency. To determine pH sensor accuracy, we compared the online pH readings from the optical sensor with offline measurements from a calibrated Nova BioProfile 400 system (Nova Biomedical). We tested a 6–7.6 pH range by using 1N HCl and 1M NaOH to adjust pH values of the proprietary medium. We tested DO sensor accuracy by comparing online values from the optical sensor to the air percentage setting of the shake flask incubator, termed “offline” in the figures. The percentage was changed through the mixed gas inlet by variation of the CO₂ percentage in the incubator. A two-point calibration at 0% and 100% was achieved by sparging air into phosphate buffered saline (PBS) with and without sodium sulfite.

k_LaStudies: We determined oxygen mass transfer coefficients (k_La) using a proprietary chemically-defined medium in 250-mL shake flasks. We used JMP software to design a full- factorial design of experiments (DoE) with varying working volumes (40, 80, and 120 mL) and shaker speeds (100, 150, and 200 rpm). Each condition was executed in duplicate, and the shake flasks were on a platform shaker with an orbital diameter of 19 mm.

We evaluated various k_La measurement methods (data not shown), and chose a method on the basis of consistency and data reproducibility. Briefly, a medium-filled shake flask was sparged with nitrogen gas (N₂) until the medium was depleted of oxygen. This medium was transferred to an empty shake flask, which was then placed on the SFR and orbitally agitated in a 37°C incubator to monitor the DO increase until saturation was reached. We calculated kLa values using the linear portion of the online DO data according to the equation ln(C^* – C)=k_La(t), in which k_La is the oxygen transfer coefficient, t is time, C is the oxygen concentration, and C^* is the saturated oxygen concentration. We analyzed and graphed results in JMP.

Cell Culture, Equipment, and Assays: We used a suspension Chinese hamster ovary (CHO) cell line from Genentech, Inc. that expresses a therapeutic monoclonal antibody, (MAb A or MAb B) to show culture performance differences in shake flasks and bioreactors. We used a CHO cell line expressing a different monoclonal antibody (MAb C) for the bolus base addition and simulated sampling experiments. Those cell lines are derived from a dihydrofolate minus (DHFR-) host that uses DHFR/methotrexate selection (10). We thawed cells and maintained them in a proprietary selective chemically defined medium for two to three weeks. We transferred cultures to a proprietary, nonselective, chemically defined medium for a two-stage inoculum train stage before entering the production stage using the same medium. A day 3 feed consisted of 20% w/v of a proprietary chemically defined feed media.

We used a Forma Scientific incubator (Thermo Scientific) and an Innova platform shaker (New Brunswick Scientific) with an orbital diameter of 19 mm for shake-flask cultures. Culture conditions were 37°C with 5% CO2 and 85% humidity. Agitation occurred through orbitally shaking of the 250-mL shake flasks at 150 rpm. Applikon 2-L bioreactors controlled with a DeltaV BioNet system (Broadley-James) produced MAb A and MAb B. Sparging with air, oxygen, or a combination through an open pipe controlled DO, and CO₂ and/or 1M sodium carbonate additions controlled pH. The initial temperature was 37°C, with a shift to 35°C on day 3; pH set-point was 7.0; and agitation of the pitch-blade impeller was 275 rpm. We used a Vicell AS (Beckman Coulter) system to obtain viable cell counts and the Nova BioProfile 400 unit for offline pH sampling.

Bolus Base Addition for pH Control: After bolus base addition, we monitored pH profiles to determine how long pH could maintain at the postbolus addition level in shake flasks. The culture used was a day 3 production culture and had a low pH of 6.6. We tested three conditions using 250-mL shake flasks with 100-mL working volume. The control flask was not pH-adjusted, one flask was adjusted to pH 7.0 and one flask to pH 7.3. We monitored pH profiles over 24 hours with a 15-second sampling interval. We used 1M sodium carbonate for the pH adjustments, and calculated the base addition amount with Equation 1 (11). V_add is the sodium carbonate volume to add; V_total is the total culture volume; PCO2 is the partial pressure of CO2 in the culture; k_H is the Henry's Law constant for CO₂ (value at 37°C used, 36 (L atm)/mol). [HCO₃–]_add is the carbonate concentration in the liquid base; pH_f is the final pH; pH_i is the initial pH; and pK_a is the first dissociation constant of sodium carbonate base (value used was 6.3).

Simulated Sampling Procedure: Using four 250-mL shake flasks, we evaluated the effect of a simulated shake flask sampling procedure on pH and DO profiles. The control flask contained media only. Cultures of various viable cell densities (1×10⁶/mL, 5×10⁶/mL, and 10×10⁶/mL) seeded the other three flasks. Agitation was stopped after 10 minutes of shaking at 150 rpm in the incubator, and we left the incubator door open to simulate manual sampling. Thirty minutes later, the incubator door was closed and agitation returned to 150 rpm. Monitoring of both pH and DO took place during this time period.