Whatever the feed stream – be it from a cell culture, waste stream from a fruit or other plant pressing, or the whey from a dairy process, it has always been a requirement that particulates be removed before application of the material to the first purification column.  This is because the particulates can clog the frits and solid phase packed into the column.  For small volumes, the solution has been to either centrifuge or filter the particulates away.  In some cases both techniques are used.

As processes are scaled up, the feed stream volumes increase and the associated issues with both centrifugation and filtration increase.  The equipment for both is expensive – especially in regulated cGMP environments and filters need to be integrity-tested before use – a time consuming process in its own right.

With COVID-19, supply chain issues have also meant that the availability of filters and (for centrifuges) spare-parts has become an issue.  Eliminate the clarification step and associated issues with supply chain and spares are also eliminated.

In the pre-column clarification steps (either filtration or centrifugation) there is an inherent variability in the clarification efficiency.  This is unavoidable and stems from the feed stream itself as the number of particulates per unit volume will vary between process runs, and from the fact that valuable target material will get trapped in the pellet or filter cake.

The only way to completely remove this variability is to remove the clarification step.

In a SMART Chromatography™ based purification process, the feed stream contains all of the particulates and these will pass through the packed column bed.  Frit materials are designed to allow these particulates to pass unhindered into the solid phase.  The solid phase itself is made from large beads, with interstitial channels allowing the particulates to traverse through the column, whilst the target molecule will bind selectively to the ligands covalently attached to the beads.

Since the early 1990s, it has been well-established that RFC provides seamless linear scalability.

As with all commonly used packed-bed purification processes, SMART Chromatography™ works best when the bed height (or in the case of RFC, bed height) remains constant.  It is for this reason that even the R&D SMART Chromatography columns are available in bed lengths that are identical to those used in the manufacturing environment.

Linear scalability leads to process predictability – reducing process development times, as the process is already known and validated during the R&D phase. The purpose of scale-up is simply to purify more target molecule per production run.

In traditional AFC purification processes, target recovery from what is applied to the column is generally quite high – in excess of 95%.  However, this recovery percentage is not entirely accurate, as it is not 95% of what was available before the feed stream was applied to the capture column.  If the clarification steps result in a loss of 15% of target, then maximum recovery (at 95% recovery on the capture column) will be just under 81%.  At 30% loss, maximum recovery is 66% and at 50% loss the recovery is 47.5%.  Processes that are apparently efficient quickly become economically unviable.

In SMART Chromatography™, the capture efficiency (or product recovery) will be similar to that for an AFC column – so 95% or higher, provided that the capture modality is identical.  However, given that loss of target is zero as all of the target material is applied to the column, the recovery is quantitative.

One of the most common issues in downstream processing is the loss of biological activity of the target molecules.  Product degradation can be causes by many factors, including enzymatic degradation, chemical and physical environment.  In cell-based systems, such as those commonly used in the manufacture of biologic therapeutics, such degradation is closely linked to the time it takes to properly prepare the feed stream prior to application of this to the capture column.

In a SMART Chromatography™-based process, it is possible to apply the feed stream directly from the bioreactor to the capture column without modification*.  This means that the time-dependent degradation simply does not occur.  Once the target molecule has been captured on the column and degradation causing contaminants have either passed through the column or have been washed away, such degradation is prevented.

(*) Some cell systems do not secrete the target molecule into the bioreactor solution and in such cases the cells need to be ruptured in order to release the target molecule into the solution prior to application of the feed stream.  This is necessary in both traditional and SMART Chromatography™ based processes.

By eliminating the requirement for clarification of the feed stream prior to application to the SMART Chromatography™ column, there is the immediate reduction of environmental impact due to the associated impact from the manufacturing of the centrifuge, filtration system and consumables.  Additional reductions may also be obtained by avoidance of feed stream preparation (for example pH adjustment).

If one also considers reductions obtained from shorter processing times (energy costs, facility and personnel costs), then the overall environmental impact can be reduced significantly compared to traditional processes.

Linear scalability also leads to a lower overall environmental impact.  Process development times are reduced, leading to lower volumes of feed stream and reagents required for the process development stages. Linear scalability also means that waste streams (excess buffer requirements, consumables, etc.) are more predictable.  Less waste can also result in lower transport costs or post-production treatments.

In processes where the target molecule is purified from what previously would have been considered a waste stream (for example, vegetable extract residue or milk whey), it is more difficult to evaluate the environmental impact – although it would be reasonable to expect it to be lower if the resulting effluent is closer to water once production is completed.

In the preceding sections, it has been discussed how SMART Chromatography™ can improve product recovery and biological activity, which naturally leads to a direct reduction in process costs.  By eliminating the capital expenditure (CapEx) on expensive centrifuge and filtration systems, there is also the continuous operational cost of filters.  The associated time and overhead costs are also eliminated.

What might not be immediately obvious is the potential reduction in costs from manufacturing unit operations not normally associated with purification.  If product recovery from the bioreactor is increased, then the volumes of material required to produce a given amount of the target molecule can be decreased and it will still be possible to obtain the desired amount of target molecule. For certain cell culture systems, the cost of the cell culture materials are very high. If recovery is improved by 30% (so an increase from 66% in the example shown above to 95%), then bioreactor volume can be decreased – an immediate impact on the process cost with every single production run.

There is usually a high degree of focus on the cost of the solid phase and the overall process cost is often held up against this. By assessing the entire cost of manufacturing from start of the cell culture process to the stage at which a purified protein is obtained (after elution from the SMART Chromatography™ column), the cost reduction will be far more than that just associated with using a different solid phase.