RESULTS
Fed-batch
Figure 1: Number of viable cells in fed-batch and concentrated fed-batch runs. (ALL FIGURES ARE COURTESY OF THE AUTHORS)
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The results of the fed-batch culture shown in Figures 1–6 are typical of such cultures. There was rapid loss of viability
following a growth phase (see Figures 1, 2), continued increase in product concentration after the culture began to decline
(see figure 3) and accumulation of waste products (lactate, see Figure 4), accompanied by a steady decline in glucose concentrations
and DO (see Figures 5, 6). Supplementing the fed batch culture four times with nutrient media during the course of the culture
resulted in a peak cell concentration of 5.8x106 cells/mL at 191 h (see Figure 1). As expected, the highest product concentration was somewhat delayed, reaching 0.4 g/L,
at 260 h (see Figure 3).
First CFB run
Figure 2: Percent of viable cells in fed-batch and concentrated fed-batch runs.
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The benefits of addition of fresh medium while removing waste media is also illustrated in Figures 1–6. pH was set by the
process without the need for a separate pH control (see Figure 7). The perfusion rate in this run varied between 0.8 and 1.5
vv/d (see Figure 8). The first run was less than ideal. The weight measurement for the Wave bag and associated control loop
to keep the level constant in the bag had a problem resulting in a working volume that was too high. A manual adjustment was
made by increasing the filtrate flow rapidly to remove this excess media. However, this adjustment was too fast for the ATF
System's cleaning action and the essentially dead-end filtration performed led to filter failure. Viability started to decline
early in the run, at <150 h, and the viable cell count also leveled off at about the same time (see Figures 1,2). The perfusion
was ended at 239 h and in spite of the dificulties, a peak cell concentration of 16.4x106 cells/mL, was reached at 195 h (see Figure 1), whereas the highest product concentration, 1.9 g/L, was reached at 239 h
(see Figure 3).
Second CFB run
Figure 3: Product concentration in fed-batch and concentrated fed-batch runs.
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The second CFB run with the ATF System differed somewhat from the first CFB run. It was performed with a more rapid increase
in perfusion rate to about twice the final rate of the first run. The perfusion rate in this run varied between 0.8 and 2.5
vv/d (see Figure 8). The cell growth was sustained longer than in the first run, achieving a significantly higher cell concentration.
The highest cell concentration, 34.5x106 cells/mL, was reached at 284 h (see Figure 1), whereas the highest product concentration, 3.8 g/L, was reached at 332 h
(see Figure 3). The viability of the culture was sustained above 85% for the duration of the culture (see Figure 2). After
an initial adjustment, glucose and lactate were sustained at about constant levels for most of the culture duration (see Figures
4,5).
DISCUSSION
Figure 4: Lactate concentration in fed-batch and concentrated fed-batch runs.
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The results in Table I show that cell, and more importantly, product concentration can be greatly enhanced with the ATF System
CFB process. Even in the first CFB run which was a less than optimal culture, nearly three–fold higher cell concentration
was achieved and more than a 4–fold increase in product concentration was obtained than in fed-batch, in about the same time
period. The results improved further in the second CFB run, increasing product concentration to nearly ten–fold. Media feed
rate appears to have had a great affect on cell growth rate and product levels. Further enhancement would be possible with
enrichment of oxygen transfer and media development.
Figure 5: Glucose concentration in fed-batch and concentrated fed-batch runs.
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Despite the increases in product concentration in CFB over fed batch, the ultimate productivity and the economical aspects
of both process modes need to be assessed and understood. Some considerations are reactor volume capacity, and the amount
of product needed, as well as the time taken to produce the amount of product.
Figure 6: Dissolved oxygen (DO) in fed-batch and concentrated fed-batch runs.
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Using the data obtained in these experiments, fed-batch is further compared to CFB. Three production scenarios, (Case A, B
and C in Table II), are offered for producing 3.8 g of hIgG, which was achieved in the second ATF run, and 100 g of hIgG.
Figure 7: pH in fed-batch and concentrated fed-batch runs.
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Table II can be used to evaluate requirements for production of a specified amount of product and to further analyze whether
those requirements correspond to a user's needs or capabilities. For example, if the user has 1-L reactors, but wishes to
produce 4 g of product, then it will require over 3 months and 9 batches to do so, using the fed-batch mode (Case B), but
will only need 1 batch and two weeks in concentrated fed-batch mode (Case C). Given the risks of running 9 batches versus
1, and the 3 month extra overhead cost, there is a significant advantage in adopting concentrated fed-batch, at the cost of
spending as little as 2.5 times more on media.
Figure 8: Flow rate in concentrated fed-batch runs.
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The relationship above is obviously relevant for any size reactor: If the user has a 100-L reactor, perhaps a single use bioreactor,
and needs 400 g of product, it will require about 10 batches and four months with a fed batch process (Case B). The user may
consider buying a new 1000-L reactor, which would do the job in one batch, but will also require associated accessories, downstream
equipment and facility space. Alternately, the user may use the ATF System, which will deliver the 400 g required in one batch
in the existing 100-L (Case C).
Table I: Summary of results.
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If the user has three reactors, 25-L, 100-L, and a 250-L to choose from, and 100 g of product is needed, then the 250-L fed-batch
option would seem to come out slightly better than the 100 L fed-batch, with a longer production cycle, or the 25-L CFB system
with a similar production timeframe but costlier media use. However, when considering seed expansion for this 250-L reactor,
which is not factored into the examples in Table II, it appears that the 25-L and 100-L reactors are needed anyway; therefore,
when assessing risk versus the gain in using the 25-, 100- or 250-L system, it appears to be similar in each case. Other factors
such as downstream processing of 25-L versus 250-L harvest need also to be taken into account.
Table II: Production of human Immunoglobulin G (hIgG) using fed-batch and concentrated fed-batch.
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In yet another scenario: What if the user has several 25-L reactors and only one 250-L reactor but wishes to produce multiple
products in a limited time? Clearly the concentrated fed-batch approach can most readily achieve that goal. The multiple 25-L
reactors can be used simultaneously, rather than waiting for the main reactor to become free. The 25-L reactors will also
not be tied up as seeds for the 250-L reactor.
CONCLUSION
The use of the ATF System to achieve exceptionally high productivity when used in a concentrated fed-batch process lends itself
to more efficient transition to upstream operation. Additionally, in the case of a midsize or smaller facility, the requirement
of expanding capacity through reactor scale-up is removed. The ATF System allows existing equipment to be more flexibly adjusted
to increased production requirements.
Because the ATF System can also be used to produce high density, large volume cell banks in disposable bags, which are used
to inoculate small or even pilot sized reactors directly from the freezer, the facility can be further optimized. Similarly,
for seed expansion, the ATF system can reduce the reactor train lengths, freeing up existing reactors for other production
requirements.
For a facility using or planning to use disposable reactors, the ability to reassign capacity to new projects could be very
valuable in reducing costs and risks in biomanufacturing.
John Bonham-Carter* is vice-president, Jerry Shevitz is president, and Edi Eliezer is vice-president, all at Refine Technology LLC, Pine Brook, NJ, jbonhamcarter@refinetech.com
. Jan Weegar and Antti Nieminen are both senior scientists at Biovian, Turku, Finland.
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