6


Figure 4. Productivity (left) and capacity utilisation (right) as a function of the linear flow rate for batch (blue symbols) and CaptureSMB (red symbols) processes.


In analogy to the abovementioned procedure, operating parameters were determined also for maximum feed flow rates of 150 cm/h, 450 cm/h and 600 cm/h.


The UV signals recorded at the outlet of each column are shown in Figure 3 (bottom) for a run with a maximum feed flow rate of 300 cm/h. The disconnected phases (61 mL length) and interconnected phases (41 mL length) are clearly distinguishable. During column loading, a rapid increase and the reaching of a plateau value of the UV signal can be observed. The plateau UV signal corresponds to the unbound impurities that are flowing through. During the interconnected phase the breakthrough of mAb from the upstream column is visible as rise from the impurity plateau level. At the same time, a rise of the UV profile is not visible at the UV of the downstream column, which indicates that the entire mAb that breaks through is adsorbed in the downstream column.


Process control based on the breakthrough UV-signals [2] is straightforward to implement and have been validated for twin-column CaptureSMB.


As reference processes, batch chromatography runs with equal resin volume (0.5 i.D. x 20 cm L column) were carried out with a load corresponding to 90% of the 1% breakthrough value [3], loading flow velocities of 150 cm/h, 300 cm/h, 450 cm/h and 600 cm/h, respectively, and the same recovery and regeneration protocol (Table 1).


The results for the maximum feed flow rates of 150 cm/h to 600 cm/h showed comparable purities of batch chromatography and CaptureSMB. (Batch runs: 2.0-3.0% aggregates, 7000-12’000 ng HCP/ mg mAb, 1.0-5.0 ng DNA / mg mAb. CaptureSMB runs: 1.5-2.5% aggregates, 5000-12000 ng HCP/ mg mAb, 2.0-4.0 ng DNA / mg mAb).


Figure 4a shows the productivity as a function of the flow rate for the CaptureSMB and batch processes. The productivity of twin-column Capture SMB is larger than the productivity of batch single column capture for any feed flow rate. This is due to two reasons: Firstly, the load of the CaptureSMB process is larger (the columns are loaded far beyond 1% DBC). Secondly, the recovery and regeneration protocol is carried out twice as fast, since the bed height during recovery and regeneration is only half the bed height (10 cm) of the batch reference run (20 cm). It can be further observed that the productivity difference with respect to batch chromatography increases with increasing flow rate (Figure 4a).


This can be attributed to the fact that a broadening of the breakthrough curves as a consequence of increasing flow rate has less effect on CaptureSMB than on batch chromatography since in CaptureSMB material that breaks through earlier due to increased feed flow rate can be likewise captured in the second column.


Figure 5. Buffer consumption as a function of the linear flow rate for batch (blue symbols) and CaptureSMB (red symbols) processes.


Therefore the load reduction required to obtain an acceptable yield (preferably > 90%) is much smaller for CaptureSMB compared to batch chromatography leading to a far less dramatic decrease in capacity utilisation (Figure 4b).


Since the load decrease (and capacity utilisation decrease) with increasing feed flow rates is less relevant for CaptureSMB, the buffer consumption remains almost constant for CaptureSMB while it increases for batch chromatography (Figure 5).


Conclusions


The CaptureSMB process offers significant performance advantages in comparison to batch chromatography. The productivity of CaptureSMB in the investigated mAb capture case was on average 35% larger while the capacity utilisation increase was 25%, which translates into 25% resin costs reduction. The buffer consumption was reduced by 20% on average. The advantages of CaptureSMB with respect to batch chromatography are resin-dependent. Preliminary studies indicate that, in the case of resins with broader breakthrough curves (i.e. larger particles) and larger static capacities, the advantages of CaptureSMB can exceed 50% increase in productivity, 40% resin cost reduction and 40% buffer savings at high feed flow rates (600 cm/h).


Due its cyclic nature CaptureSMB is very well suited for combination with continuous upstream production such as perfusion fermentation. By continuous manufacturing the equipment size (pumps, columns) can be typically reduced by at least one order of magnitude.


References


1. Müller-Späth, T.A., Monica; Baur, Daniel; Lievrouw, Roel ; Lissens, Geert; Ströhlein, Guido; Bavand, Michael; Morbidelli, Massimo; , Increasing Capacity Utilization in Protein A Chromatography. Biopharm International, 2013. 26(10): p. 33-38.


2. Warikoo, V., et al., Integrated continuous production of recombinant therapeutic proteins. Biotechnology and Bioengineering, 2012. 109(12): p. 3018-3029.


3. Mahajan, E., A. George, and B. Wolk, Improving affinity chromatography resin efficiency using semi- continuous chromatography. Journal of Chromatography A, 2012. 1227: p. 154-162.


Read, Share and Comment on this Article, visit: www.labmate-online.com/articles How a LC Purification System can be Adapted for Different tasks and still be Affordable


This question was the starting point for developing a new type of preparative liquid chromatography equipment for purification tasks. Contichrom® process equipment from Knauer successfully combines the flexibility of batch HPLC systems with the efficiency and throughput of continuous chromatography systems.


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Instead of requiring five different systems this can be achieved with a single equipment and control software. Typical application areas for Contichrom range from FPLC separations of proteins to HPLC separations of pharmaceutical active ingredients.


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The MCSGP process principle works with two columns rather than one. Through continuous column switching, impure side fractions of the target peak can be recycled and separated again, maximising extraction of the product. Yield and purity can be increased by 50%, while achieving up to 10-times higher throughput and a 70% reduction in solvent or buffer consumption.


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LAB ASIA - JANUARY/FEBRUARY 2014


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