Shear stress investigations on an oil-water emulsion system result in drop breakup over the experimental time. To count drop sizes in pumping processes, the suitability of inline endoscopy was successfully demonstrated in this protocol.
Pumps are mainly used when transferring sterile culture broths in biopharmaceutical and biotechnological production processes. However, during the pumping process shear forces occur which can lead to qualitative and/or quantitative product loss. To calculate the mechanical stress with limited experimental expense, an oil-water emulsion system was used, whose suitability was demonstrated for drop size detections in bioreactors1. As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes need to be counted over the experimental time of shear stress investigations. In previous studies, the inline endoscopy has been shown to be an accurate and reliable measurement technique for drop size detections in liquid/liquid dispersions. The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. In order to express the drop size, the Sauter mean diameter d32 was used as the representative diameter of drops in the oil-water emulsion. The results showed low variation in the Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating the reliability of the measurement technique.
Pumps are used to transfer cell cultures in the pharmaceutical and biotechnological industries. During the pumping process, mechanical stress can result in irreversible cell damage, which might impair the quantity and quality of the product1-4. The level of mechanical stress depends on the pump type and pump settings, as demonstrated in previous studies5-6. Commonly, peristaltic, syringe and diaphragm pumps are used for single-use (SU) technology based applications. These pumps result in high local shear forces caused by the compression of the pump tubing and the pulsating flow7.
In order to overcome these drawbacks, magnetically levitated centrifugal pumps (MagLev centrifugal pumps) constitute a promising alternative. The motor is magnetically driven in order to avoid narrow gaps between the impeller and the pump housing (Figure 1). A previous study investigated the MagLev centrifugal pumps and showed lower mechanical stress in Chinese Hamster Ovary (CHO) cells compared with peristaltic and 4-piston diaphragm pumps5. In addition, hemolysis analyses revealed no significant blood trauma and thrombus formation over a range of operation conditions using these pumps8-11. The findings demonstrate that use of these specifically designed pumps applies less mechanical stress on biological systems in comparison with peristaltic and diaphragm pumps. To investigate the mechanical stress with limited experimental expense, an oil-water emulsion model system is recommended due to its cost- (ca. 99.8%) and time-reduced (ca. 99.5%) application compared with biological cell culture systems.
As drop breakup of the oil-water emulsion system is a function of mechanical stress, drop sizes must be counted over the experimental time of shear stress investigations. Many techniques for sizing drops are available, which can be divided into sound, laser and photo based techniques12. In particular, the use of the photo-optical probe inline endoscopy shows almost identical drop sizes for manual and automatic detections (standard deviation below 10%) and enables a detection of 250 drops per minute13. Because of its accuracy and reliability, the endoscope technique has been shown to be an effective standard measurement technique for drop size distributions in liquid/liquid dispersions when compared with other commonly used probes (e.g., fiber optical forward-backward-ratio (FBR) sensor, focused beam reflectance method (FBRM) and the two-dimensional optical reflectance measurement technique (2D-ORM))12,14. Moreover, the suitability of inline endoscopy for measuring drop sizes in a stirred vessel has been demonstrated several times in previous investigations15-18.
Based on a prior study6, this protocol describes the use of inline endoscopy to determine drop sizes (Sauter mean diameter) of an oil-water emulsion system in pumps. The Sauter mean diameter was used as a comparison criterion in order to estimate the mechanical stress of the multi-use (MU) MagLev centrifugal pumps, a peristaltic and a single-use (SU) 4-piston diaphragm pump.
Figure 1. Magnetically levitated centrifugal pump-system. (A) The principle of a bearingless motor and (B) the PuraLev 200MU are shown as an example. Please click here to view a larger version of this figure.
The aim of this protocol is to show the suitability of the inline endoscopy technique for drop size measurements in pumping processes. For this purpose, drop sizes of an oil-water emulsion system were determined and a measured Sauter mean diameter was calculated to characterize the mechanical stress of the MagLev centrifugal pumps as well as their counterparts, a peristaltic and a 4-piston diaphragm pump. The results showed low variation of the measured Sauter mean diameters, which were quantified by standard deviations of below 15%, indicating that drop sizes have been reliably and accurately measured. As a consequence, the measured Sauter mean diameter could successfully be used as a comparison criterion to evaluate the mechanical stress of the pumps investigated. The MagLev centrifugal pumps revealed larger measured Sauter mean diameters, indicating lower mechanical stresses on emulsion drops compared with the peristaltic and 4-piston diaphragm pumps. In studies to date, inline endoscopy has been shown to be a robust and simple technique for reliable drop size measurement1,6,12-14,20-21, which was also confirmed by this study. In comparison to alternative measurement techniques, such as the fiber optical FBR sensor, the FBRM and the 2D-ORM technique, the endoscope technique can be used as the standard method for obtaining precise data in liquid/liquid applications12,14.
The easy handling of the inline endoscopy and the simple production of the non-biological oil-water emulsion system enables a straightforward procedure for drop size detections according to the protocol text (see above). Nevertheless, it should be mentioned that the position of the endoscope probe depends on the fluid flow in the storage vessel. Further investigations (data not shown) have revealed that the lens of the probe should be located directly beneath the inlet tube for lower flow rates up to 5 L min-1 in order to avoid a multiple detection of one drop19. For sharp images at flow rates over 5 L min-1, it is recommended to position the probe at least 10 cm away from the inlet tube. Independent of process parameters, the holder of the inline endoscopy should be stable in order to avoid a shifting of the probe, which can result in blurred images.
Furthermore, it should particularly be noted that the drop size detected is close to the lower detection limit of the applied photo-optical system, where the minimum detectable drop diameter is 6.5 µm. As the manufacturer-provided software has been improved, inline endoscopy techniques can reliably detect a minimal drop size of 1 µm. Moreover, the image processing will be further developed to enable online monitoring of industrial applications.
While the present study focused on relatively low flow rates of up to 3.4 L min-1, future studies should consider a wider range of operation conditions. First investigations have been carried out at flow rates up to 20 L min-1 (data not shown). However, a 1:2 dilution (csurfactant = 0.09 ml L-1, coil = 0.64 ml L-1) of the oil-water emulsion system is recommended at flow rates over 10 L min-1 19, as increased drop breakup caused by higher mechanical stress would otherwise affect drop detection and reduce the number of drops detected. Tests were carried out with a 1:2 dilution and compared with results of an undiluted oil-water emulsion system. For both approaches, the Sauter mean diameters have been reliably measured (standard deviation below 5%). Therefore, reduced volume fraction (1:2 dilution) did not influence the measured Sauter mean diameters, and thus a drop-drop breakup was negligible.
These powerful experimental approaches provide a good basis for the improvement of the endoscopy technique as well as the related image acquisition, recognition and result analyzer software. Furthermore, the suitability of the endoscopy technique to classify pump types and series according to their mechanical stress was successfully demonstrated. The results obtained are essential for pump design development and the optimization of pumps to reduce cell damage.
The authors have nothing to disclose.
The authors would like to thank the Commission for Technology and Innovation (CTI, Switzerland) for their financial support (No. 13236.1 PFFLI-LS).
CCD camera | Allied Vision Technologies GmbH | GX2750 | Equipment for inline endoscopy |
C-Flex Biopharmaceutical Tubing | Saint-Gobain Performance Plastics | 374-375-4 | Tube Select a tubing length of about 45 cm before the pump. |
C-Flex Biopharmaceutical Tubing | Saint-Gobain Performance Plastics | 374-375-3 | Tube Select a tubing length of about 45 cm after the pump and clamp on the flow sensor to this tubing. |
CLAVE Connector | Victus | 011-C2000 | Sampling port |
Controller LPC-200.1-02 | Levitronix GmbH | 100-30030 | PuraLev 200MU controller |
Controller LPC-600.1-02 | Levitronix GmbH | 100-30033 | PuraLev 600MU controller |
LeviFlow Clamp-On Sensor LFSC-12 | Levitronix GmbH | 100-30329 | Flow sensor for flow rates below 5 L min-1 |
LeviFlow Converter LFC-1C-CS | Levitronix GmbH | 100-30328 | Flow sensor output device |
Masterflex I/P Easy Load | Fisher Scientific AG | EW-77963-10 | Peristaltic pump |
Mitos free flow valve | Parker Hannifin Europe Sàrl | FFLQR16S6S6AM | Valve |
Mobil Eal Arctic | Exxon Mobil Corporation | Mobil EAL Arctic 22 | Oil Prepare the emulsion directly before the experiment. |
Motor | Elektromotorenwerk Brienz AG | 7WAC72N4THTF | Motor for agitator shaft |
Motor BSM-1.4 | Levitronix GmbH | 100-10005 | PuraLev 200MU motor |
Motor LPM-600.4 | Levitronix GmbH | 100-10038 | PuraLev 600MU motor |
Norm-Ject 10 mL Luer Lock | Restek Corporation | 22775 | Syringe |
Pump Head LPP-200.5 | Levitronix GmbH | 100-90525 | PuraLev 200MU pump head |
Pump Head LPP-600.18 | Levitronix GmbH | 100-90548 | PuraLev 600MU pump head |
Quattroflow 1200-SU | Almatechnik AG | QF 1200 | 4-piston diaphragm pump |
SciPres Sensor | SciLog | 080-695PSX | Pressure sensor |
SciPres Sensor Monitor | SciLog | 080-690 | Pressure sensor output device |
SOPAT-VF Inline Endoscopic Probe | SOPAT GmbH | Inline endoscopy | |
Stroboscope | Drello GmbH & Co KG | Drelloscop 255-01 | Equipment for inline endoscopy |
Triton X-100 | Sigma-Aldrich | X100 | Surfactant Handle with gloves and goggles. (acute toxicity, eye irritation) |