An experiment was run to identify the influence o f the presence o f cell debris on the diafiltration performance (%T). Lysate was spun down to remove the spheroplasts and cell debris and the supernatant was used as the feed in constant volume diafiltration. Contrary to experiments with whole lysate, the level o f %T was still above 50% after 1 hour o f operation (Figure 6.13). This result rules out the possibility that %T might be going down solely as a result o f a decrease in the concentration driving force, as pointed out in section 6.2.3.
A further aspect that is interesting to note is that in this case there would be no detrimental effect o f a different ionic strength or pH. If indeed the protein molecules increase in volume at a lower ionic strength, this only appears to affect the %T when in the presence o f cell debris. One possible explanation for that might be that with the whole lysate a secondary layer composed o f cell debris and proteins forms on the surface o f the membrane (Le and Atkinson, 1985), resulting in a reduced effective molecular weight cut-off.
Van Reis et al. (1997) claim that protein aggregation is concentration dependent, since it is a result o f protein-protein interactions. If one assumes that it can also be induced by the presence o f cell debris, this might explain the non-availability o f some protein during the diafiltration o f lysate. This “non-available” protein would become increasingly important as the “available” protein is removed, as opposed to diafiltration o f lysate supernatant, where aggregation would not occur due to the absence o f debris. Also in the case o f total permeate recycle the overall protein concentration is constant and so the proportion o f “non-available” protein would remain unchanged and small throughout the process. This could explain why %T is less than 100% in the total recycle experiment.
Figure 6.14 does show that when spun down lysate is used, the totality o f the antibody fragment present in the retentate side is “available” for transmission, since the plot o f Cp vs. Cr crosses the origin. The decrease in %T as a result o f fouling can be seen from an enlargement o f this figure in the lower concentration range (Figure 6.15). The experimental points arch slightly, showing the gradient is lower nearer the origin.
100
80
60
40
20
040
50
010
20
30
60
Time (min)
Figure 6.13 Effect o f diafiltration processing time on percentage transmission o f antibody fragment, experiment done with spun down lysate. Error bars are the propagated error from the standard deviation as a result o f two to fo u r dilutions o f
each permeate and retentate sample. An exponential decay curve was assumed (% iT
=
99^-00091^ ^2 ^ 0.59). Experiments conducted under constant retentate flo w rate
(average velocity over membrane 0.4 m s'^). TMP varied between 0.07 and 0.14 bar during the experiments and the flu x was kept constant at 35 Lm'^h'^. The volume o f spun down lysate (feed) was 0.5 L.
80
70
G)E
c
o
50
(0U 40
o8 30
(0 0)E
k. 2 0 o Q.60
2 040
80
0Retentate concentration (mgL
Figure 6.14 Permeate concentration (Cp) as a function o f retentate concentration (Cr) during diafiltration o f spun-down lysate. Error bars represent the standard deviation as a result o f two to fo u r dilutions o f each permeate and retentate sample. A least squares linear decay f i t was assumed: Cp = 0.78 Cr + 0.7, r^ = 0.995 calculated with all the points (— ) or Cp = 0.96 Cr - 0.3, r^ = 0.999 calculated excluding the p o in t fo r higher concentration (corresponding to t=3 minutes in Figure 6.13) as the value o f Cp may have been underestimated due to initial dilution ( ----). Experiments conducted under constant retentate flo w rate (average velocity over membrane 0.4 m s'^). TMP varied between 0.07 and 0.14 bar during the experiments and the flu x was kept constant at 35 Lm'^K^. The volume o f spun down lysate (feed) was 0.5 L.
O)
E
c
o
*3 (0 L. 4-"c
o oc
o
o OS 0)E
k. o Û. 64
2 0 0 24
6Retentate concentration (mgL‘^)
Figure 6.15 Permeate concentration (Cp) as a function o f retentate concentration (Cr) during diafiltration o f spun-down lysate (enlargement o f Figure 6.14 fo r the lower range o f concentrations). Error bars represent the standard deviation as a result o f two to fo u r dilutions o f each permeate and retentate sample. The straight line represents the least squares linear decay f i t represented in Figure 6.14 by (---) but adjusted to cross the origin: Cp = 0.96 Cr.
6.3 Conclusions
Several conclusions can be made from this set o f experiments. First o f all the percentage transmission presents a very fast decay during diafiltration o f E. coli lysate. To compensate for the low performance, the membrane area required for the separation needs to be large, so that the process can be conducted within a reasonable period o f time. This is detrimental from an economic point o f view, particularly in a disposable process, where the membrane has to be replaced at each new batch.
The main contribution to the decrease in transmission cannot be exclusively attributed to fouling since the decrease is not observed as strongly with total permeate recycle. In fact the transmission appears to drop as a result o f a decrease in the concentration o f the species being removed from the system, possibly that o f Fab’. A possible explanation is that there may be an amount o f product that is not available for transmission. This “non-available” material could be in the form o f aggregates assayable with ELISA, but not present in the spun down material, since a %T decay is not observed in this case. Another possibility would be adsorption o f Fab’ to the cell debris.
The hypothesis that the reduction in %T is due to a decrease in the concentration driving force does appear unlikely since it does not occur with spun down lysate. It could also be that a proportion o f Fab’ swells due to a change in the environment, but this would have to be an irreversible effect, since the quantity o f “non-available” Fab’ is apparently constant with time, as can be hinted from Figure 6.9. To confirm this fully, experiments can be done where the diafiltration buffer has a higher ionic strength or a pH close to 8.3, which is the isoelectric point o f the Fab’ antibody fragment. If swelling does occur then the effect should be apparent in the spun down lysate experiments, unless there is less hindrance to the passage o f the swollen proteins (no secondary membrane formed by the cell debris).
Some fouling occurs during diafiltration o f spun-down lysate, and in this case it should be the sole mechanism responsible for the decrease in %T. Intuitively one might expect the fouling to be more severe in the presence o f cell debris, but these may also form a cake that prevents the formation o f a protein layer, thereby improving the overall transmission, as Kuberkar and Davis (1999) observed when yeast is added to a
BSA solution. This also highlights the need for more membrane filtration studies with real process streams instead o f idealised protein solutions.
One further reason for the results observed could be that material might get released from the cell debris during centrifugation o f the samples, but this is unlikely due to negligible level o f shear damage induced by a micro-centrifiige (Boychyn, 2000). The freeze/thawing o f the samples prior to analysis could potentially also lead to the release o f more Fab’, that would still have been intracellular at the time o f the MF experiment. This is however unlikely to have a strong impact, since three hours o f periplasmic release at 60°C allow the release o f more than 95% o f the Fab’ available (Bowering,
2000).
The observation that some material may not be “available” for filtration is important and may go some way to explaining other results reported in the literature. The fact that even small amounts o f “non-available” material can lead to such significant drops in apparent transmission means that any membrane optimisation procedure needs to be approached with care. This result is also crucial in determining how best to use the membranes in a disposable fashion as a true understanding o f transmission is essential in knowing when to cease diafiltration on economic grounds.
Where transmission decay is unavoidable and due to fouling new approaches will need to be developed to minimise its effects. A strategy for membrane area optimisation will be described in Chapter 7, followed by experimental evaluation in Chapter 8.