Tips & Tricks we have picked up during our work with 0.5 liter P-SUBs
On this page randomly organized with Tips & Tricks for:
- The Drive Unit Clotho
- Software for the Drive Units really makes the difference
- P-SUB's being CellMembra and CellRetention both 500 ml - more or less identical to 250 and 300 ml VV
We strongly recommend reading the User Manuals
Remember that mammalian cells line behave different to different conditions. Some cell do not like alternating flow and some does. Some cells don't like re-circulating flow and some does.
----------------------------------
Variables for CellMembra-500 connected to Clio CM80 O-SUP
Circulated volume, SP, Set Point: 0 - 500 ml/min
Stroke volume, 100 % stroke/systole, ml: ~50
Number of strokes/min: SP / 50 + C cycle time
Cleaning cycle initiates when pressure limit inside pump is detected.
Cleaning Velocity, CV, m/sec: 0 to >10
Cycle time, ms: 0.05-0.1
Harvest Velocity, HV, m/sec: 0 – 1.5 depending on SP
First run - wait with inoculating cells until you are familiar with the setup
Run a CellMembra sterile with no cells, perhaps even / only sterile water and learn how the setup work for a day or two! Helps further to insure the HFF is prepared.
With re-circulated water get data of velocities and volumes, play with the wide range of within you can alter Drive Unit programming
Be confident with a pumped broth volume along, tangential to, axial of the HFF like one SUB Working Volume / min – see the process works before you reduce pumped volume
Work with the buttons Vacuumize / Pressurize and see for yourself that SUB liquid surface jumps up and down in the range of 10 mm
Be sure when the automatic Cleaning cycle takes place the SUB volume jumps up and down
Be sure you get a sufficient amount of permeate out.
Cleaning cycle is like a concentrate of well-known ATF and will look somewhat rough
When you are confident …………go with cells …. see if cell viability at all is reduced as to planned “high” pumped volume
----------------------------------------------
Variables for CellRetention-500 connected to Thalia TM80 A-SUP
Exchanging volume, SP, Set Point: 0 – 1000 ml/min
Single 100% stroke volume, systole, ml: ~50
One reciprocating stroke volume, systole + diastole, ml: ~100
Number of reciprocating strokes, exchanging strokes/min: SP / 100 = BpM
Velocity, velocity peak for both systole + diastole, m/sec: 0 - >10
First run - wait with inoculating cells until you are familiar with the setup
Run the CellRetention sterile with no cells, perhaps even / only sterile water and learn how the setup work for a day or two!
With water get data of velocities and volumes, play with the wide range of within you can alter Drive Unit programming
Be confident with a pumped broth volume along, tangential to, axial of the HFF like one SUB Working Volume / min – see the process works before you reduce pumped volume
Work with the buttons Vacuumize / Pressurize and see for yourself that SUB broth liquid surface jumps up and down in the range of 10 mm
When you are confident …………go with cells …. see if cell viability at all is reduced as to planned “high” pumped volume
-------------------------------------
Fouling problems - insure sufficient velocities
At endurance use with too low pumped broth volumes and few cleaning cycles with high mammalian cell density may easily foul the HFF and cells will fast accumulate also in the valve body
Clotho Drive Gas pressure is limited to maximum 1.2 bar pressure hereby protecting the HFF
If you experience blocked HFF. Stop the process. Use the two Vacuumize / Pressurize bottoms on Drive Units monitor and manually force the diaphragm to perform full strokes. Hope to flush out debris and open up the HFF. Perform this process repeatedly. You should be able to see through the transparent dome that the diaphragm moves between both end points fast. If the flow though the HFF is possible.
If you have installed a permeate pump able to generate harvest fluid pressure in opposite direction. Back flus pulse may be helpful in deposit removal.
------------------------
During use of HFF
The process may create symptoms of membrane or straw, lumen fouling. Typically, the process have not been properly optimized:
Flow path occlusion: Lumen diameter may be too small to handle high suspended solids concentration. Select different process or larger straw diameter
Concentration factor too high or membrane surface area is not adequate to process the feedstock volume. Please retest membrane capacity calculations at small scale.
Operating conditions such as process TMP and cross flow rate are not optimal.
--------------------------------
Important around Clotho Drive Unit
As to the internally pre-set max operating pressure of 1.2 Bar (19 psi) its difficult to break the HFF, which generally handles 2 Bar (30 psi).
Be sure you have read the manual !
If you don't have the Clotho hardware version with easily software up-gradable with a USB memory stick - ask for a free exchange
-------------------------
Operating HFF
TMP is the difference between the average upstream, feedstock pressure and permeate pressure of a Cross-Flow-Filter. It is the driving force for membrane flux.
TMP refers to Trans-Membrane-Pressure. TMP is the pressure that moves liquid and low molecular weight solute through the membrane. When controlling a filtration process with TMP control, pressure is regulated, controlled at both the feedstock inlet and retentate exit port, and flux (flow through the membrane) is governed by porosity, pore size, area, viscosity, solute concentration, concentration polarization and pressure. In this mode, flux is high at the beginning of the process, but diminishes as a function of time/throughput.
Using Flux Control, the permeate flow rate is kept constant using either a permeate control and/or permeate pump characteristics. Typically, the permeate flow rate starts lower than with TMP control, but may offer a faster overall filtration process by preventing premature fouling of the membrane. The rate of filter fouling is reduced due to less aggressive conditions minimizing the gel layer. TMP is allowed to rise slowly as a function of time/throughput. The best way to run with flux control is to use a pump set to a conservative flow rate depending upon the feedstock solute concentration. Or based on programmable permeate pump characteristics according to the feedstock pump characteristics.
Aeration
With open end 6 mm tube and 90% Oxygen its possible to reach 80 mio/CHO cell/ml with good viability at 300 rpm pumping down.
The mini is selectable available with high efficient OD 6 x 10 mm micro sparger - though care combined with media composition (Serum) is need avoiding foam.
---------------------------------------------------
Setup
Similar to above for CellMembra. Difference being that Thalia A-SUP exchange double the volume compared to the Clio O-SUP pumped volume. The Drive Unit Clotho selectable gives both options.
-------------------------------------------
END