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ATF-manufacturing
Overview of ATF-manufacturing production technology
 Refine Technology recommends a production platform called ATF-manufacturing. Combining multiple different process improvements and enhancements in areas such as cell banking through to product harvest and beyond, this production platform gives you the following benefits:
- Reduced risk by allowing a delayed decision to build a facility
- Reduced facility capital cost (smaller facility required for same product Kg output)
- Reduced validation costs
- Reduced cost of goods
- Reduced open handling of cells
- Reduced manual operation
- Reduced contamination risks
- Fewer unit operations
- Less equipment required and less variation in equipment
- More reproducible cell expansion and productivity
The platform integrates perfectly with other innovations, such as the FlexFactory™ from XCellerex or Pharmadule’s pre-build offering, but can equally be used in traditionally designed facilities with either steel or stand-alone single-use equipment.
The unit operations and applications where the ATF-manufacturing platform can offer significant improvement include:
- ATF-cellbanking
- ATF-seedexpansion
- ATF-cellcultivation
- ATF-mediumexchange
- ATF-filtration
- ATF-diafiltration
- ATF-Vconcentration
- ATF-seedexpansion
- ATF-cellconcentration
- ATF-clarification
- ATF-Pconcentration
- ATF-virusremoval
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Upstream: CELL BANKING AND SEED EXPANSION
ATF-cellbanking, ATF-seedexpansion
 Cell banking is normally a rather laborious manual process using centrifuges and a series of concentration, separation and resuspension operations, subject to much variariability. The cell bank produced is generally vials containing 1ml or 10ml of cell suspension of similar concentration (and viability). Since this cell bank creation takes several hours (or up to a day depending on the number of vials), it is impossible to expect the first and last vial to be the same.
The cells in these vials are critical since they are the first step in the manufacturing process, whether for small scale research purposes or for eventual inoculation to a 20,000L reactor. Generally for manufacturing, several vials will be thawed and a series of cell expansions will occur in parallel so that a choice of the best from them will be used to inoculate the first bioreactor. This is unavoidable due to the inherent variation caused by the inefficient manual process that created the cell bank.
There is risk, uncertainty and variation in these steps and there is a significant time involved.
ATF-cellbanking solves all the problems with the existing procedures. This is done by ensuring the cells always stay in a healthy environment within the sterile reactor, for example they never suffer stress through a lack of oxygen, centrifugation or being starved of nutrients. The media exchanges and freezing fluid introduction all occur rapidly inside the reactor and the cell bank is transferred to disposable bags without any open handling nor individual manual testing of cell concentrations. The entire process can be automated and completed within an hour: the first cell bag banked will be identical to the last – and this can be validated.
ATF-seedexpansion removes the variation from the first passages from cell bank to bioreactor. The cell bank can inoculate directly a small bioreactor from the freezer without the need for centrifugation and DMSO removal in advance. No open handling is needed. The bioreactor can be from 5L to 50L or larger depending on your facility (disposable or traditional), and the expansion of the cells occurs in concentrated fed-batch mode to ultra-high cell densities, e.g. 70-150m/ml. This first seed reactor can then inoculate a large reactor (20-50x) without intervening scale-up steps, saving a significant amount of time and capital and reducing manufacturing risk for each batch. The larger reactors can then be operated in standard batch or fed-batch mode, or further productivity improvements can be made by implementing concentrated perfusion or concentrated fed-batch.
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CELL RETENTION APPLICATIONS IN CELL CULTIVATION
Overview of Perfusion, Concentrated Perfusion and Concentrated Fed-Batch CFB™
 Recirculating hollow fiber systems have been used for a long time with mixed results. The recurring problem
with these and all other cell retention systems is the difficulty in scaling up and staying sterile. Many cell
retention systems scale up based upon surface area, or a separation chamber of sorts, that upon transfer into a
manufacturing environment become very challenging to run efficiently or reliably. Many companies who use
internal spin filters or centrifuges, for example, typically invest a considerable amount of time optimizing their
often unique installations.
Trying to avoid cell damage or time outside of the reactor in a dead zone are common issues with those
systems. These problems are eliminated when the ATF System is used as a production platform. The ATF System scales
nearly linearly and is simple and quick to install, operate, validate and maintain. With the ATF System process development
becomes simple since there is little equipment optimization required so all time can be
spent improving media and bioreactor conditions. The ATF System allows significant cell density to
be achieved rapidly.
Key benefits include:
- Low shear
- Cells in constant equilibrium with reactor contents
- Gentle separation of aggregates
Some cell lines show a decrease in cell size, allowing a faster molecular transfer across the cell wall
(increase of surface area to volume ratio). Together these benefits deliver high viability and higher
growth rate and thereby lead to a surprising high cell concentration in almost every cell line tested.
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Perfusion
Perfusion had become regarded as a process option to be used when the stability of the protein is low or if
the expression of the cell-line is minimal such that a constant output becomes more economic. Perfusion has
suffered from complex or unreliable equipment diminishing its attractiveness, and attracted a perception
that process development or operation is difficult.
No longer, you’ll be glad to know! The ATF System solves all these problems and make the process as simple as -
some would say simpler than - fed-batch processes.
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Concentrated Perfusion
Perfusion is dead – Long live Concentrated Perfusion!
The death of perfusion has always been more of a marketing success than anything else. Several blockbuster and
medium size marketed drugs are made today in perfusion processes – and more are planned. Today however,
perfusion is very much on the rise with the ATF System leading the way. So what is concentrated perfusion
and how it can improve on the existing perfusion methods?
Key Concentrated Perfusion characteristics:
- Ultra-high cell densities, in the region of 70-200m/ml
- Cell viabilities of 90% and higher
- Requires 1-3 vv / day media (not more)
- Single cell suspension, no aggregates
- Same simple process control as perfusion – but higher productivity
- Can be used to produce cell density only (without protein or virus) – for example for use in ATF-cellbanking
Concentrated Perfusion is only possible with the ATF System, other systems such as cell settlers, hydrocyclones, spin-filters or sonoperfusion cannot achieve the same results.
When should perfusion be used and not Concentrated Perfusion? Generally, when the product quality is affected
by cell density, or when there is some inhibitory molecule that would require an unreasonably high media
flow to remove, e.g. limit to 15m/ml.
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CFB: Concentrated Fed-Batch
This is an exciting platform technology for cell cultivation. It is a highly versatile process which permits great increases in cell and product concentrations as compared to Fed Batch (FB). FB is widespread in the biopharma: it’s an established platform and relatively repeatable.
However, challenges in the industry (such as competitive products for the same indication or desired COG
reductions) are forcing many to explore new options. Concentrated Fed-Batch is one such option now gaining
ground within the industry with several companies scaling up the process.
The CFB process uses the ATF System which simultaneously nourishes the culture and concentrates the product
within the bioreactor. Protein product titers of 17g/L in CHO and even higher in other cell lines have been reported. Further improvements in titer are expected as process optimization continues.
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Downstream: CELL RETENTION APPLICATIONS IN HARVEST OR SEPARATION OPERATIONS
Cell Concentration, Rapid Medium Exchange
 Cell viability is critical for these applications where the quality or expression of the product could be affected by slow or damaging processing. The ATF System provides a significant benefit over other equipment such as TFF, depth filtration or centrifuges due to its low shear and ability to filter material fast. Often an operator would be happy with a 3-4 hour step for concentrating 20m cells to 200m at 50L for example.
The ATF System simplifies the development of the manufacturing process by streamlining the operation, achieving results 3-4 times faster, generally reducing the manual handling of cells and so minimizing risks at all times. Normally, both the capital costs and the overall cost of ownership are reduced. For example, a 200L single use bioreactor can be concentrated to 20L in an hour assuming cell densities of 20-50m/ml, while ensuring cell viability does not drop.
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Downstream: GENERAL FILTRATION AND SEPARATION OPERATIONS
Clarification, Protein Concentration, Diafiltration, Virus removal, Virus Concentration
The ATF System has started to be used as a replacement for many other types of filtration equipment. Depending on
conditions, the results can be extremely good, and we advise you to contact us prior to choosing which ATF
System would be best for your application.
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MICROCARRIERS
Wash, Concentration, Perfusion, Rapid Medium Exchange, Trypsinisation, Wash, Cell-Microcarrier Separation,
Virus Perfusion, Virus Harvest.
 Microcarrier processes are inherently more complex and more involved than concentrated fed-batch or fed-batch
processes. The extra steps required for preparation of the vessel and various washing procedures during the
process often means that there is significant manual intervention and multiple points of potential contamination.
The ATF System simplifies and makes reliable almost every complex step in an adherent cell-line process which
uses microcarriers such as Cytodex. From washing the carriers post-sterilisation, to perfusion, to
cell-microcarrier separations and the final harvest, the ATF System offers significant benefits. The
benefits arise due to a specially designed single-use 70µ screen module filter which is inserted into the
ATF System housing allowing it to operate as an efficient macro-filter. The same filter is used throughout the
process, while the ATF System is fully automatic for all process steps at almost all flows. The ATF
System for microcarrier processes scales from 1L to 5000L and is also compatible with all single-use bioreactor types
on the market today.
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FILTER CHOICES
 Which filter is best for which application? Which pore size, which lumen diameter? Is PES better than PS?
There are many applications of the ATF System and each may require a different filter. Generally there is one particular type recommended for each application which will give the best results. Occasionally, however, it may be better to use another variation so please ask us to help with this decision.
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