Results and discussion
Experimental validation
The complete integrated continuous downstream process was run on
laboratory scale with the process setup presented in Figure 2. If the
downstream process were connected to a perfusion bioreactor, the load
concentration would vary over time. Therefore, in order to test the
performance of the control strategies, an external disturbance was
introduced in the form of a 20% decrease in the load concentration, to
emulate a change in the harvest concentration that would occur during a
perfusion run.
The chromatogram shown in Figure 6 corresponds to a run with 9 cycles,
including an initial loading stage for process startup. After startup,
steady product output was achieved by cycle 4, after running the process
with a cycle load volume of 36 mL. The reference value obtained for the
product output used in the loading control was 23.1 mg. The load
concentration was reduced by 20% at the beginning of cycle 4, and the
effects in the final product were seen after a further two cycles due to
the residence time in the virus inactivation loop, as well as the delay
between the capture and polishing steps. The measured product output
decreased to 18.0 mg, and as this value differed from the reference
value, the loading controller provided a new cycle load volume
proportional to the decrease in concentration. This led to a gradual
increase in the cycle load volume from 36 mL in cycle 5, to 44.6 mL in
cycle 9, which resulted in an increase in product output until a value
similar to that of the reference value was reached. In fact, the product
output had already reached 22.3 mg only one cycle after the disturbance
was detected in the pool of the final product. This means that the
product output had almost reached its original value approximately 2
hours after the disturbance was detected in the final product, thus
confirming the effective implementation of the loading controller with a
rapid response. In a typical perfusion run, a change in concentration of
20% would be made over a much longer time, thus giving sufficient time
for the controller to adjust the cycle load volume to maintain the
desired product output. The pooling strategy was also successfully
validated, since the absorbance cutoff for the end of product pooling in
the polishing step was adjusted according to the peak height, as shown
in Figure 7.
The effect of the controller on the response of the downstream process
is shown in Figure 8. At the new steady state following the disturbance,
implementing the controller led to an increase in the product output of
21.8% per cycle (Case c1A) compared to the process without the
controller (Case c1B), while the cycle load volume increased by 23.9%.
The process was run without any disturbance (Case c0) to provide a
baseline to compare the process response with and without the
disturbance.
An external disturbance in the load flow rate was considered, but not
investigated experimentally. The reason for this was that a change in
flow rate would not affect the process as the load volume was
continuously tracked by the control software Orbit. In other words, if
the flow rate was reduced, the cycle time would be extended, but the
cycle load volume would be unaffected, thus having no impact on the
product output.
Process performance
indicators
Table 1 presents the values of several process performance indicators at
steady state for the 4 scenarios studied. The batch process, which was
run at a load volume of 45 mL to achieve the same resin utilization as
in the continuous process, had a yield of 68.3%, which is significantly
lower than the 87.5% obtained in the continuous process (Case c0). This
is because there is no interconnection between the columns during the
loading phase of the capture step, which leads to a higher product loss
due to breakthrough. The product output was similar in both cases (22.6
mg in Case “Batch”, compared to 23.5 mg in Case c0) since the total
amount of product loaded onto the capture column was the same.
Therefore, the specific buffer consumption is also close to the value
obtained in the continuous process (16.7 mL mg-1compared to 16.0 mL mg-1 in Case c0). Three parameters
affect the productivity: the product output, the process time and the
total volume of resin. The higher the product output and the lower the
process time and the resin volume, the higher the productivity. On the
one hand, the process time in the batch process was higher (240 min
compared to 95 min) due to the fact that the loading phase and the
recovery phases were not carried out simultaneously, unlike in the PCC
operation. On the other hand, the resin volume in the batch process was
6 mL (1 mL of capture resin + 5 mL of polishing resin), which was less
than in the continuous process (8 mL) because, in this process, 3
columns were used in the capture step due to the PCC operation. In this
case, the difference in the process time had a much greater effect than
the difference in the resin volume, which led to a significantly lower
productivity in the batch process (22.6 mg day-1mL-1) in comparison to the continuous process (44.1 mg
day-1 mL-1), as can be seen in Table
1.
In an evaluation of the effect of the controller on the performance
indicators, it was found that the amount of purified product per cycle
was higher in the process with control (Case c1A) than in the process
without control (Case c1B). This, in turn, led to an increase in resin
utilization of 24% (from 71.4% to 87.9%), as estimated from the
breakthrough curve, as more product was purified in each cycle compared
to the process without the controller. In addition, the resin
utilization and the product output in Case c1A had similar values to
those of the process with no external disturbance. Another effect of the
higher product output per cycle was the decrease in the specific buffer
consumption, i.e., the volume of buffer consumed to purify a certain
mass of product. The reduction in specific buffer consumption was
estimated to be 21% compared to the process without the controller. The
productivity was lower in Cases c1A and c1B than in Case c0, as the
concentration decreased, leading to a lower amount of product being
purified per unit time. Regarding the yield, 87.5% of the incoming
protein was recovered in the final product in the case without
disturbance. When the load concentration was decreased, the yield was
not negatively affected, as the yield was 87.2% in Case c1A and 85.1%
in Case c1B. If the disturbance had been a concentration increase
instead, the control system would have reduced the PCC cycle length
until the minimum cycle time was reached (79 min), and the yield would
have been similar. In contrast, in the process without control, the
cycle length would have remained the same, and the yield in the loading
of the capture step would have decreased significantly.
The purity was analyzed at different stages of the process in Case c0 to
ensure product quality, and the results are given in the Supplementary
Material, where it can be seen the purity of the product increased after
each chromatography step. The purity of the final product was 99.1%.
The main impurities were present in the breakthrough of the capture
step, as shown in the chromatograms corresponding to the supernatant
(Figure S1 in Supplementary Material), the capture pool (Figure S4), and
the capture breakthrough (Figure S3). A significant amount of impurities
was also present in the breakthrough of the polishing step (Figure S5).
The chromatogram of the pure product (Figure S6) did not contain any of
the peaks corresponding to the detergent (Figure S2), thus showing that
the detergent was effectively removed in the process. Product purity was
not measured in Case c1A, but the only difference between Case c0 and
c1A was the change in the load concentration, while the impurity profile
was the same. It is therefore reasonable to assume that the purity would
have remained unchanged in Case c1A.