Tips & Tricks Perfusion-SUB 500 ml

Tips & Tricks we have picked up during our work with 500 ml P-SUBs

On this page randomly organised with Tips & Tricks for:

  • The 2 Drive Unit sisters: Clotho and Lachesis. Mechanically and electronically are more or less alike.
  • Software for the Drive Units are more or less identical
  • P-SUB's being CellMembra and CellRetention both 500 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.

This T&T file updated: 2018-05-15

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Variables for CellMembra-500 integrating Clio-M80

  • Circulated volume, SP, Set Point: 0 - 650 ml/min
  • Stroke volume, 100 % stroke/systole, ml: ~35
  • Number of strokes/min: SP / 35 + C cycle time
  • Cleaning / Harvest cycles, C/H ratio: 1 – 25
  • 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

Variables for CellMembra-medium integrating Clio-100

  • Circulated volume, SP, Set Point: 0 – 1,300 ml/min
  • Stroke volume, 100 % stroke/systole, ml: ~100
  • Number of strokes/min: SP / 100 + C cycle time
  • Cleaning / Harvest cycles, C/H ratio: 1 – 25
  • Cleaning Velocity, max CV, m/sec: ~10
  • C cycle time, ms: 0.05-0.1
  • Harvest Velocity, max 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 cleaning/harvest ratio of like 1:10 - if not tighter like 1:5
  • Only with tight cleaning/harvest ratio you can reduce circulated volume – you could go to 1:2
  • 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

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.

Prep of HFF

  • Ultra-Filtration (UF) membranes are soaked with Glycerine, Glycerol. In particular Hollow-Fiber-Filter Ultra-Filtration membranes (1 kDa to 750 kDa). These filters require alcohol/water solution treatment to restore wetting / glycerol removal. Typical 25/75 ethanol /water solution
  • Most fresh virgin membranes need to be prepare for use. Forgetting membrane wetting by media and or absorbents leads to an increase in mass transfer resistance.
  • All HFF manufacturer gives some easy to access and good recommendations to prepare the HFF for use. See the Spectrum PDF files at bottom.
  • Micro-Filtration (MF) HFF is per standard supplied by PerfuseCell. MF HFF is from manufacturer and PerfuseCell supplied dry. Insure that no trapped air is locked in the HFF cartridge. Be sure to recirculate media, buffer, clean water under pressure to diffuse trapped air into the water. During re-circulation, close the permeate and gradually close the retentate valve until the absolute pressure within the cartridge housing is max at 1.2 Bar (15-20 psi).

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.

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Variables for CellRetention-500 integrating Thalia-M80 A-SUP

  • Exchanging volume, SP, Set Point: 0 – 1,300 ml/min
  • Single 100% stroke volume, systole, ml: ~35
  • One reciprocating stroke volume, systole + diastole, ml: ~70
  • Number of reciprocating strokes, exchanging strokes/min: SP / 70 = 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

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.
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