Technical Background
The ATF™ System was originally designed for perfusion processes in mammalian cell culture, using hollow fiber filters to achieve an efficient cell separation with low shear and allowing robust large scale manufacturing. The filtration system was taken forward quickly as a preferred cell retention device and now is used in several large scale commercial perfusion facilities to manufacture proteins and antibodies.
The standard bioreactor types of glass in R&D and steel in pilot and production are easy to get an ATF System retrofitted to, with a range of standard adaptors and ports fitting all fermenter suppliers' equipment, including Sartorius /Braun BBI, Applikon, New Brunswick, Bioengineering, Biolafitte/Pierre Guerin etc.
The rapid adoption of single use systems, first as rocker and Wave type systems, then as stirred tank variations both in small rigid plastic configurations and the larger scale bags, required new single use connectors to allow the addition of Refine's cell retention and perfusion device. Connectors such as the Pall Kleenpack, GE's DAC and the Opta from Sartorius Stedim all perform well to achieve this. Today, ATF Systems have been connected to almost every type of single use bioreactor (SUB) from 1L to 1000L volumes: Sartorius Cultibag and STR, XCellerex, Hyclone, GE Wave, ATMI, NBS Celligen Blu and Millipore CellReady.
Refine has been offering several different types of filters for the ATF System for the past 10 years, but recently two versions, a hollow fiber filter at 30-50Kda pore size and a screen module at 70um have proved exceptionally popular:
The 30-50Kda ultrafiltration module allows retention of proteins in the bioreactor changing a perfusion process into a concentrated fed-batch process with exceptionally high cell concentrations and yields in a single harvest - suitable for rapid replacement of the standard fed-batch platform.
The 70um screen module is suitable for adherent cells and microcarrier based processes. Vaccine processes, some based upon MDCK or Vero cells, have proved particularly well suited for improvement using the ATF System. In the last few years, the rise of larger scale stem cell culture has shown new process challenges. Stem cells are often grown on microcarriers and can therefore benefit from the ease the ATF System brings to the multiple wash steps and cell-microcarrier separation steps required. Concentration of stem cells can also be performed rapidly with low shear.
ATF System Technology
Shown here are a controller and the pump head of an ATF4 System. The ATF4 pump head has been cut open so that the movement of the diaphragm is visible. This system would typically be attached to a 10L bioreactor.
The controller first directs a controlled flow of pressurized air into the bottom of the ATF pump and forces the diaphragm to move upwards. When the diaphragm reaches the top, the expansion is detected, and the controller swaps to a vacuum source to bring the diaphragm down again. This cycle is repeated every 5-10 seconds in each direction, giving a total cycle time of approximately 10-20 seconds.
The large surface area of the diaphragm minimizes shear as the diaphragm moves. The ATF4 pump head displaces about 400ml of liquid with each exchange, which means this slow movement generates a fast flow of about 3-4L per minute.
The ability for the ATF System to generate high flows with low shear has many benefits for the culture, including gentle separation of aggregates, cleaning of the filter surface and support for extremely high cell concentrations such as 100-200m cells per milliliter in many different cell line types including CHO and PER.C6®
This is a simplified animation to explain what is happening inside an ATF System when attached to a reactor. It shows a traditional steel reactor with a connection using two bivalves to enable a re-sterilisable connection. However, single use bioreactors are just as easy to connect up to, using single use connectors such as Pall's Kleenpack or GE's ReadyMate.
- Pressurized air flows into the bottom of the ATF pump
- The air pressure and flow forces the diaphragm gently up, creating a fast flow of cells and media up through the system
- There is rapid flow through the fibers, with a filtrate outflow showing on the right (for harvest, clarification, concentration etc.)
- The flow of media and cells continues through the system and into the bioreactor
- The system then reverses: air is exhausted through the pump bottom
- The diaphragm moves down creating a rapid flow from the reactor through ATF System, ready for the next cycle.
- The cycle repeats approximately every 5 seconds
A unique feature of the ATF System action is its inherent ability to reduce filter fouling. The combination of a reversible flow through the filter with alternating high and low pressure cycles results not only in efficient tangential flow but also in significant trans-membrane fluxes with back-flushing action on each cycle. While the back-flushing volumes may be small it is sufficient to keep the filter from fouling even at very high cell concentration, greater than 200 x106 cells/ml in some cases. The ATF System action allows the use of smaller filters than would otherwise be expected.
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