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 product .
the level of mechanical stress depends on the pump type and pump settings , as demonstrated in previous studies .
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 flow . 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 pumps .
in addition , hemolysis analyses revealed no significant blood trauma and thrombus formation over a range of operation conditions using these pumps .
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 .
99.5% ) 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 techniques .
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 minute . 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 ) ) .
moreover , the suitability of inline endoscopy for measuring drop sizes in a stirred vessel has been demonstrated several times in previous investigations .
based on a prior study , 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 .
( a ) the principle of a bearingless motor and ( b ) the puralev  200mu are shown as an example .
the investigations were performed using a piped pump setup ( figure  2 ) , which enables mechanical stress experiments at flow rates up to 60  l  min and pressure drops up to 2  bar to be performed . as shown in figure  2 , the experimental setup is comprised of the storage vessel , the pump circuit , and the equipment for the inline endoscopy technique .
peripheral elements were integrated into the closed loop to monitor the flow rate v and pressure drop p at different pump settings .
ensure that the bioreactor ( d  =  0.15  m , h / d  =  2.2 ) is equipped with an impeller for the dissolution of the surfactant and make sure that the inlet tube dips into the fluid to avoid a gas entering .
equip the pump loop with a syringe port , the investigated pump , a clamp - on flow meter , a single - use pressure sensor and a hand - wheel valve .
after connection of the pump loop to the storage vessel , connect the pump head to the motor and prepare the endoscope probe .
mount the changeable reflection plane , a rhodium mirror in this case , at the probe tip and adjust the distance between mirror and lens to 150  m .
connect the probe to the stroboscope via a fiber - optic cable and the camera of the endoscope to the computer via an ethernet cable . then , connect the camera and the stroboscope together via a trigger - box cable .
start the computer and open the manufacturer - provided software , which includes an image acquisition and recognition software , as well as result analyzer software .
click on the button  detect device  in the upper left corner of the screen to detect the camera . under 
directory settings  select the location on the computer for saving images and activate the command  create trigger sub folders  . enter the process parameters in section  trigger mode : ready  .
frame rate : 7.5  hz frames per trigger : 50 number of triggers : 60 trigger interval : 60  sec after completion of all preparatory work , pour 5  l deionized water into the storage vessel and switch on the pump to fill the pump and the pump loop .
switch off the pump and add 0.9 ml of surfactant ( csurfactant  =  0.18  ml l , surfactant,20c  =  1,070  kg  m , critical micelle concentration ( cmc ) : cmc    0.018  ml l , surfactant    10    cmc ) with a 10 ml pipette under stirring .
position the endoscope probe so that the lens is located directly beneath the inlet tube . set the flow rate of 3.4  l  min and the pressure drop of 0.03 , 0.3 or 0.61  bar by varying the impeller speed and the hand - wheel valve .
weigh 6.3  g of oil directly in the syringe ( oil  =  1.26  g  l , oil,20c  =  989.5  kg  m ) . start the image acquisition software and add the oil via the syringe port .
after 1 hr , finish the shear stress investigation and clean the inline endoscope as well as the bioreactor with the integrated pump loop .
subsequently , prepare the experimental setup for the next pumping process . open the automatic image recognition software in the main menu . under
 batch root directory  select the location on the computer for saving the files ( all*.csv ) . select the column 
image series path  and click on the button  add image series subfolders  in the lower left corner of the screen to load the image series .
select the column  search settings ( * .pss or auftrag_*.mat )  and click on the button  set search settings  in the lower - middle of the screen to load process parameters in order to specify the drop recognition .
select the column  search pattern ( * .psp or f_*.mat ) and click on the button  set search pattern  in the lower right corner of the screen to load the process parameters in order to specify the drop analysis . start the image recognition by clicking on the button  start batch  .
after completion of image recognition , express the detected drop sizes by the sauter mean diameter ( d32 ) , or any other representative mean value or distribution of choice by using the result analyzer software .
load folder(s )  in the upper left corner of the screen to load the previously saved all*.csv file .
select the relevant value ( e.g. , sauter mean diameter ) in the drop - down list in the upper middle of the screen to visualize the results . for the calculation of the diameter enter the scaling of 0.6591  m pixel on the right , which is provided by the manufacturer .
pump circuit for piped pump setup using inline endoscopy as the measurement technique : ( 1 ) storage vessel , ( 2 ) syringe port , ( 3 ) pump , ( 4 ) pressure sensor , ( 5 ) flow sensor , ( 6 ) stroboscope , ( 7 ) computer with the manufacturer - provided software , and ( 8) endoscope probe .
ensure that the bioreactor ( d  =  0.15  m , h / d  =  2.2 ) is equipped with an impeller for the dissolution of the surfactant and make sure that the inlet tube dips into the fluid to avoid a gas entering . equip the pump loop with a syringe port , the investigated pump , a clamp - on flow meter , a single - use pressure sensor and a hand - wheel valve .
after connection of the pump loop to the storage vessel , connect the pump head to the motor and prepare the endoscope probe .
mount the changeable reflection plane , a rhodium mirror in this case , at the probe tip and adjust the distance between mirror and lens to 150  m .
connect the probe to the stroboscope via a fiber - optic cable and the camera of the endoscope to the computer via an ethernet cable .
then , connect the camera and the stroboscope together via a trigger - box cable . start the computer and open the manufacturer - provided software , which includes an image acquisition and recognition software , as well as result analyzer software .
click on the button  detect device  in the upper left corner of the screen to detect the camera . under 
directory settings  select the location on the computer for saving images and activate the command  create trigger sub folders  . enter the process parameters in section  trigger mode : ready  .
frame rate : 7.5  hz frames per trigger : 50 number of triggers : 60 trigger interval : 60  sec after completion of all preparatory work , pour 5  l deionized water into the storage vessel and switch on the pump to fill the pump and the pump loop .
switch off the pump and add 0.9 ml of surfactant ( csurfactant  =  0.18  ml l , surfactant,20c  =  1,070  kg  m , critical micelle concentration ( cmc ) : cmc    0.018  ml l , surfactant    10    cmc ) with a 10 ml pipette under stirring .
position the endoscope probe so that the lens is located directly beneath the inlet tube . set the flow rate of 3.4  l  min and the pressure drop of 0.03 , 0.3 or 0.61  bar by varying the impeller speed and the hand - wheel valve .
weigh 6.3  g of oil directly in the syringe ( oil  =  1.26  g  l , oil,20c  =  989.5  kg  m ) .
after 1 hr , finish the shear stress investigation and clean the inline endoscope as well as the bioreactor with the integrated pump loop .
 batch root directory  select the location on the computer for saving the files ( all*.csv ) . select the column 
add image series subfolders  in the lower left corner of the screen to load the image series .
select the column  search settings ( * .pss or auftrag_*.mat )  and click on the button  set search settings  in the lower - middle of the screen to load process parameters in order to specify the drop recognition .
select the column  search pattern ( * .psp or f_*.mat ) and click on the button  set search pattern  in the lower right corner of the screen to load the process parameters in order to specify the drop analysis . start the image recognition by clicking on the button  start batch  .
after completion of image recognition , express the detected drop sizes by the sauter mean diameter ( d32 ) , or any other representative mean value or distribution of choice by using the result analyzer software .
load folder(s )  in the upper left corner of the screen to load the previously saved all*.csv file .
select the relevant value ( e.g. , sauter mean diameter ) in the drop - down list in the upper middle of the screen to visualize the results . for the calculation of the diameter enter the scaling of 0.6591  m pixel on the right , which is provided by the manufacturer .
pump circuit for piped pump setup using inline endoscopy as the measurement technique : ( 1 ) storage vessel , ( 2 ) syringe port , ( 3 ) pump , ( 4 ) pressure sensor , ( 5 ) flow sensor , ( 6 ) stroboscope , ( 7 ) computer with the manufacturer - provided software , and ( 8) endoscope probe .
figure  3 shows the particle recognition images after a pumping time of 1 hr . the upper four images show the drops before recognition and the lower four images show the drops marked by the recognition software .
the drops detected are highlighted with a green edge . comparing the upper and lower images
the images on the left show the drop distribution for the maglev centrifugal pumps puralev  200mu and puralev  600mu , and those on the right show the 4-piston diaphragm and the peristaltic pump .
an optical assessment allowed an initial classification of the mechanical stress in the model emulsion system .
this revealed that larger drop sizes and lower drop counts were generated by the maglev centrifugal pumps compared with the 4-piston diaphragm and peristaltic pump .
consequently , the maglev centrifugal pumps , especially the puralev  200mu , showed reduced drop breakage , indicating lower mechanical stresses .
emulsion drops before ( a , b , c , d ) and after ( e , f , g , h ) particle recognition after 1 hr of pumping using ( a , e ) the puralev  200mu , ( b , f ) the puralev  600mu , ( c , g ) the 4-piston diaphragm pump , and ( d , h ) the peristaltic pump under identical operating conditions ( 3.4  l  min and 0.03  bar ) .
sauter mean diameter 
  further investigations revealed standard deviations below d32    0.4  m and guaranteed reproducible results when using inline endoscopy . therefore , multiple investigations were not required for this approach , which additionally reduced the experimental expense . in order to express the drop size , the sauter mean diameter d32 ( see eq . 
1 ) was used as the representative diameter of drops in the oil - water emulsion for this approach . in general , the sauter mean diameter decreased over time for all pump types and pump settings until reaching steady state .
investigations in this study confirmed the progression of the sauter mean diameter ( figure  4a to d ) , curves of the puralev  200mu ( figure  4a ) and the peristaltic pump ( figure  4d ) being exemplarily discussed in this protocol .
in contrast to the puralev  200mu , the sauter mean diameters were up to 40% smaller for the peristaltic pump at the same operation conditions ( flow rate  =  3.4  l  min ; pressure drop  =  0.03  bar ) . as a consequence , higher mechanical stresses resulted in an increased drop breakup and therefore smaller drop sizes .
furthermore , the sauter mean diameter decreased with increasing pressure drop in the puralev  200mu ( figure  4a ) , which indicated the dependency of drop size on pressure drop .
in contrast , the peristaltic pump showed a sauter mean diameter of d32,60min  =  10  m at the end of the experiment for all process parameters ( figure  4d ) .
however , the results reflect physical understanding of the drop breakup : with higher mechanical stress , smaller sauter mean diameters were determined ( see also figure  5 ) .
for each measurement point , at least 300  drops were determined in order to guarantee statistical certainty .
the maximal standard deviation decreased for the puralev  200mu from d32,4min    42  m and for the puralev  600mu from d32,6min    21  m to approximately d32    0.5  m at the end of the pumping process .
the decreased standard deviation resulted from the increased homogeneous drop size distribution until steady state was reached . in comparison to the maglev centrifugal pumps , the peristaltic and 4-piston diaphragm
typical profiles of sauter mean diameters d32 over time and the determination of the measured sauter mean diameters d32,m
. comparison of sauter mean diameters d32 ( a ) for the puralev  200mu , ( b ) for the puralev  600mu , ( c ) for the 4-piston diaphragm pump , and ( d ) for the peristaltic pump .
the sauter mean diameters d32 were determined at a flow rate of 3.4  l  min and pressure drops ranging from 0.03 to 0.61  bar .
the measured sauter mean diameter d32,m was calculated for the last 10 min  ( boundary ) .
the resulting standard deviation of the sauter mean diameters d32 ( n    300 ) is shown .
measured sauter mean diameter as a comparison system 
  as described above , the sauter mean diameter decreased over time until the drop sizes reached steady state . in the last 10  min of the experimental time ,
the average value of the sauter mean diameter was calculated to determine the measured sauter mean diameter , which was used as a comparison criterion ( see boundary of figure  4a - d ) .
the measured sauter mean diameters d32,m are shown for a flow rate of 3.4  l  min and a pressure drop range from 0.03 to 0.61  bar in figure  5 .
larger measured sauter mean diameters were determined for both maglev centrifugal pumps ( 200mu and 600mu ) and the 4-piston diaphragm pump at lower pressure drops and impeller speeds .
the peristaltic pump revealed measured sauter mean diameters of d32,m  =  10  m for all process parameters .
as previously mentioned , shear forces were independent of the pressure drop for the peristaltic pump .
the largest measured sauter mean diameters of d32,m  =  36  m for the puralev  200mu and d32,m  =  34  m for the puralev  600mu were obtained at a pressure drop of 0.03  bar . in comparison with their counterparts ,
these results indicated a lower rate of drop breakup and thus lower mechanical stress resulting from the use of the centrifugal pumps .
the standard deviation of the measured sauter mean diameters during the steady state was below 15% , thereby confirming reliable and accurate values for the drop sizes .
measured sauter mean diameters for the maglev centrifugal pumps and their counterparts at 3.4  l  min and pressure drops of 0.03 , 0.30 and 0.61  bar .
the resulting standard deviations of the measured sauter mean diameters d32,m during the steady state are shown . 

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 measurement , 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 applications .
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 in order to avoid a multiple detection of one drop . for sharp images at flow rates over 5  l  min , 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  m . as the manufacturer - provided software has been improved , inline endoscopy techniques can reliably detect a minimal drop size of 1  m .
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
first investigations have been carried out at flow rates up to 20  l  min ( data not shown ) . however , a 1:2 dilution ( csurfactant  =  0.09  ml l , coil  =  0.64  ml l ) of the oil - water emulsion system is recommended at flow rates over 10  l  min  , 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 .
wolfgang dornfeld and reto schb are employees of levitronix , ltd . , manufacturer of the maglev centrifugal pumps .
 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 . 
Introduction
Protocol
1. Experimental Setup
2. Measurement and Image Analysis
Representative Results
Discussion
Disclosures