Tips & Tricks we have picked up during our work with the 3 liter P-SUB
On these pages further various Tips & Tricks can be found here – https://perfusecell.com/perfusion-support/tips-tricks-info-aa/
The present file contains:
- Setup of Clotho
- HFF prep ATF
- HFF prep TFF
- Understanding HFF
- Use of HFF
- Variables for CellMembra-3
- Variables for CellRetention-3
- Fouling problems
- Important around Clotho
- HFF conditions
- Aeration
- Alarms
- Various
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- Setup between CellRetention and CellMembra on Clotho Drive Unit
Select up front which of the two principles is the plan. Clotho operate both principles. Difference being that Thalia A-SUP exchange double the SUP volume compared to the Clio O-SUP pumped volume.
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- HFF prep for 3 liter SUBmerged with Thalia SUP – series-31
https://perfusecell.com/perfusion-bioreactor/cellretention-atf-submerged-31
The SepraPor HFF is supplied unified with the SUB and has not been pre-wetted or flushed before installation. The complete P-SUB system is supplied dry. Wetting of the HFF is recommend insuring robust performance. Ad 2.5 liter sterile water or cell culture media to properly wet the HFF. Add the sterile wetting solution to the SUB through a suitable sterile filter. Start operating Thalia SUP by programming Clotho so the sterile wetting solution is alternated from the SUB into the SUP at app 800 ml volume per minute (2 x 400 ml for Thalia) at 16 strokes per minute. It is recommended to perform this alternating process for 5 minutes. Then sterile liquid pass perpendicular over the HFF membrane from inside lumen from the SUB. And emptied out via the permeate outlet by a suitable pump at app 150 ml/min into an external bag as waste. Process performed until all the sterile liquid has passed the HFF and removed as much sterile liquid as possible from the SUB. Stop Thalia and permeate pump. Empty the SUB via other deep tube or harvest tube. System is sterile and ready for media and inoculation.
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3. HFF prep for 3 liter Integrated SUB with Clio SUP – series-30
https://perfusecell.com/perfusion-bioreactor/cellmembra-tff-integrated-30
The SepraPor HFF is supplied unified with the SUP and has not been pre-wetted or flushed before installation. The complete P-SUB system is supplied dry. Wetting of the HFF is recommend insuring robust performance. Ad 2.5 liter sterile water or cell culture media to properly wet the HFF. Add the sterile wetting solution to the SUB through a suitable sterile filter. Start operating Thalia SUP by programming Clotho so the sterile wetting solution is alternated from the SUB into the SUP at app 400 ml volume per minute at 16 strokes per minute. It is recommended to perform this alternating process for 5 minutes. Then sterile liquid pass perpendicular over the HFF membrane from inside lumen from the SUB. And emptied out via the permeate outlet by a suitable pump at app 150 ml/min into an external bag as waste. Process performed until all the sterile liquid has passed the HFF and removed as much sterile liquid as possible from the SUB. Stop Thalia and permeate pump. Empty the SUB via other deep tube or harvest tube. System is sterile and ready for media and inoculation.
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5. Use of HFF
Hollow-Fiber-Filter are characterized by large effective surface membrane area and high packing density. However, compared to the other membrane configurations, the decrease of local flux and Trans-Membrane-Pressure (TMP) along flow direction are more prominent in the HFF configuration because of the serious internal pressure drop in the narrow diameter fiber lumen. A problem followed the pharma industry for decades.
Retentate velocity (from broth volume to HFF inlet and via HFF retentate outlet to SUP inlet)
- Recommended ml/min/lumen = 10-15 per 60 cm long HFF = 16-25 ml per 100 cm
- HFF lumen ID 1 mm then cross section = 0.0078 cm2
- Lumen volume for 600 cm = 0.0078 x 60 = 0.468 cm3 / 0.78 cm3 per 100 cm HFF
- Velocity, m/min = 16-25 ml : 0.78 = 20 to 30
- Recommended Harvest Cycle velocity, m/sec = 20 - 30 : 60 = 0.1 to 0.5
Permeate / product harvest flow in ml/min – traditionally via a peristaltic pump
- Typically 1/3 to 1/5 of SUB Working Volume / day
Cleaning velocity – sequential selected
- Reference of retentate, harvest velocity suggested 0.1 to 0.5 m/sec
- Accepted max pressure drop, DeltaP / ∆P along the HFF in mBar: 100 as example
- Clotho will when, like the 100 mBar DeltaP / ∆P is reached initiate the Cleaning Cycle sequence with the m/sec as pre-programmed – like 5 to 10 times harvest velocity – to be investigated individually
Ratio between harvest and cleaning cycles
- Highly depending on process, bio-mass concentration, media composition
- Could be 30-50 harvest cycles and 2 cleaning cycles
- Clotho will depending on programmed max DeltaP / ∆P start Cleaning Cycle
- end-user have to figure out how many and which velocity
Max DeltaP / ∆P selected
- An alarm will appear on display with 5 sec average when programmed DeltaP / ∆P is reached
- The desired max DeltaP / ∆P is heavily depending on the process, the cell line, etc
Bio-mass concentration too high or is membrane surface area is not adequate to process the feedstock volume. Please retest membrane capacity calculations at smaller scale.
Operating conditions such as process DeltaP / ∆P and cross flow rate are not optimal.
In general all PerfuseCell revolutionizing P-SUB systems are equipped with customised HFF from www.meissner.com
https://perfusecell.com/perfusion-support/membrane-filter-and-background-43/seprapor-hff-info
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- Variables for CellMembra-3 connected to Clio CM80 O-SUP working TFF – series-30
- Circulated volume, SP, Set Point: 0 – 450 ml/min
- Stroke volume, 100 % stroke/systole, ml: 50
- Number of strokes/min: SP / 100 + C cycle time
- Harvest Velocity, HV, m/sec: 0.1 – 1.5 as selected via Set Point
- Cleaning Cycle initiates when DeltaP / ∆P pressure is detected
- Cleaning Velocity, CV, m/sec: 4 to 10
First run - after HFF is ready - 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
Start with a pumped broth volume along, tangential to, axial of the HFF like 1/5 SUB Working Volume / hour – see the process works. HFF pressure drop ranging 10 to 50 mBar.
Work with the buttons Vacuumize / Pressurize and see for yourself that SUB liquid surface jumps up and down in the range of 5-10 mm
When the pre-programmed automatic Cleaning Cycle takes place the SUB volume jumps up and down. Be sure you get a sufficient and constant volume of permeate.
When you are confident …………go with cells …. see if cell viability at all is reduced as to planned “high” pumped volume
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- Variables for CellRetention-3 connected to Thalia TM80 A-SUP working ATF – series-31
- Exchanging volume, SP, Set Point: 0 – 900 ml/min
- Single 100% stroke volume, systole, ml: 50
- One reciprocating stroke volume, systole + diastole, ml: ~100
- Number of reciprocating strokes, strokes/min, BpM: SP / 18
- Harvest Velocity, HV, m/sec: 0.1 – 1.5 as selected via Set Point
- Cleaning Cycle initiates when DeltaP / ∆P pressure is detected
- Cleaning Velocity, CV, m/sec: 4 to 10
First run - after HFF is prepared - 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 within the Drive Unit programming.
Start with a pumped broth volume along, tangential to, axial of the HFF like 1/5 SUB Working Volume / hour – see the process works. HFF pressure drop ranging 10 to 50 mBar.
Work with the buttons Vacuumize / Pressurize and see for yourself that SUB broth liquid surface jumps up and down in the range of 5-10 mm.
Observe when the automatic Cleaning Cycle takes place the SUB volume jumps up and down
Keep the permeate flow constant and get a sufficient amount of permeate out as harvest
When you are confident testing with water …………go with cells ….
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- Fouling problems - insure sequential sufficient velocity
At endurance use with selected low broth / feedstock / retentate velocity (like 0.5 m/sec), permeate flow at 2,500 / 24 = 110 mL/hour reasonable mammalian cell density and no cleaning cycles – the HFF will eventually clock, foul and DeltaP / ∆P increase, an alarm occur and stop the process.
See DeltaP / ∆P development on this link - https://perfusecell.com/perfusion-support/tips-tricks-info-aa
Clotho Drive Gas pressure is limited to maximum 1.2 bar pressure hereby protecting the HFF. If you experience high DeltaP / ∆P or blocked HFF – via the alarm - stop the process. Try with the two Vacuumize / Pressurize bottoms on Clotho Drive Unit display and manually force the SUP diaphragm to perform full stroke and HFF inlet pressure. In the plan to flush out debris somewhat rough and open up the HFF lumen. Perform this process repeatedly. If the flow though the HFF is possible.
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- 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 Clotho manual !
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- HFF conditions and understanding
Hollow-Fiber-Filter is a specific defined design of the Cross-Flow-Filter (CFF) principle, HFF is a compact, circular elongated device accepted in the industry and possible to clean with correct equipment and understanding. The current HFF originates from the dialyses industry developed at MIT shortly after WWII. Opposite of a flat, box shaped flat sheet cassette which is not possible to clean. This Tips & Trick focus on 200 nm, 0.2 µm pore size membranes for mAbs expression.
The HFF consist of a number of porous straw, lumen inside a plastic tube, Lumen sealed open in each end with epoxy creating a cartridge with a permeate outlet through the plastic tube. Feedstock, broth pumped along inside the lumen and what’s allowed to pass the membrane is named permeate.
Fouling, clocking of any CFF is the normal function of separation of broth and product
TMP - (Trans-Membrane-Pressure) is the difference between the average upstream, feedstock inlet pressure and permeate pressure after the Cross-Flow-Filter membrane. The pressure difference across the filter membrane. TMP is the driving force for membrane flux = harvest. TMP will increase between Cleaning Cycles as a function of the severe fouling increase.
TMP is the net pressure that forces the part of the broth to become permeate through the membrane and is typically calculated as the average of the inlet and outlet pressures minus the permeate pressure. The equation is generally expressed as:
TMP = (P_in + P_out) / 2 ÷ P permeate
Several factors influence both TMP and pressure drop, impacting how they interrelate. In membrane filtration, membrane fouling is a significant factor that increases TMP over time. Fouling occurs when particles, colloidal materials, or biological substances accumulate on the membrane surface, increasing resistance to flow. This can lead to a higher TMP as more pressure is required to achieve the same harvest flow rate.
TMP refers to Trans-Membrane-Pressure. TMP is the driving pressure difference 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, surface area, broth 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 as to membrane fouling. This fouling is what Clotho / Thalia / Clio is able to remove via the Cleaning Cycles.
DeltaP / ∆P – (Pressure-Drop) is the difference in pressure between two points in a flow system. It reflects the resistance the fluid encounters as it moves through a filter or a series of lumens in parallel. The greater the resistance, the larger the Pressure Drop, which are caused by factors such as the length of the lumen, its diameter, the roughness of its interior surface, possible increased fouling and the viscosity of the fluid.
The pressure increase is the difference between the HFF feedstock inlet and the retentate exit port. Low, close to atmospheric pressure in the broth volume of the SUB. High at the retentate outlet as function of fouling. DeltaP ∆P will increase as a function of the ID of the lumen decreases as to unavoidable fouling. Pressure Drop are affected by the characteristics of the fluid being processed, such as its viscosity and temperature. A higher viscosity fluid, such as containing high biomass levels, will naturally result in a greater pressure drop, as will lower temperatures that increase fluid density and resistance to flow.
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 fouling layer. TMP is allowed to rise slowly as a function of time / harvest 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.
Flux rate most often measured in LMH = liters / m2 / hour
The flux / permeate / harvest flow is typically ranging 1/3 of bioreactor volume / day.
TMP as a function of flux is not constant along the lumen inner surface, but an average measure. The fouling layer inside the lumen is not constant, have similar thickness at inlet and outlet. Higher at inlet and decreasing over length of lumen. Then harvest passing the membrane is lower close to feedstock inlet and higher close to retentate outlet.
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- Aeration
The range of 3 liter P-SUBs is available with:
- Different micro sparger material and pore size (MF surface) resulting in different size bubbles - take care – media combined with Serum will create foam.
- Nano size bubbles with good transfer rate of O2 to media and good Kla values available via integrated NF surfaces …………..
With minimum bubble creation, good transfer ratio, high Oxygen concentration it’s possible to reach >100 mio/CHO cell/ml with good viability at 200 rpm agitation pumping down.
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Alarms from Clotho
DeltaP / ∆P measured constantly by Clotho. Max DeltaP / ∆P is programmable and the selectable number will be the basis of the Cleaning Cycles. DeltaP / ∆P development is an important measure and stored as needed. Clotho gives an alarm even out over 5 sec when reached.
Clotho software includes a multitude of parameter, variables. Alarms so far available for:
- DeltaP / ∆P selected ranging 10 to 400 mBar - select low numbers like 50 mBar for a start and find a suitable balance between many harvest cycles and few cleaning cycles
- Vacuum – depending of source / must be lower than 200 mBar absolute
- Pressure – fixed at 1.2 Bar
All alarms visible on Clotho display and distributed via OPC-UA to external SCADA like Lucullus.
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- Various
We strongly recommend reading various User Manuals.
Remember that mammalian cell lines do behave very different to different conditions. Some cell lines do not like alternating flow and some do. Some cells don't like re-circulating flow, and some do.
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