The Renaissance of Protein Purification - Progress in downstream processes have not kept pace with increases in upstream yields. It is time for protein purification to make a comeback. - BioPharm


The Renaissance of Protein Purification
Progress in downstream processes have not kept pace with increases in upstream yields. It is time for protein purification to make a comeback.

BioPharm International

Fortunately, the polishing area—unlike the capture area—is beginning to benefit from advances such as disposable chromatography systems, which have been implemented in a number of processes.4 In one example, a disposable 0.5-L membrane can remove process-derived impurities such as DNA, viruses, and endotoxins from a 1000-L feed stream just as well as a stainless steel column that is a hundred times larger. Results from a case study examining the economics of such systems demonstrated that disposable equipment can provide a cost savings up to 70% in MAb polishing.5 The disposable options benefit from lower capital investment and significantly reduced material costs because these small devices consume 95% less buffer. And for viral clearance, disposable chromatography offers additional advantages beyond cost savings: it eliminates the need to carry out virus carryover and cleaning validation studies.

In many processes, virus filtration (the other main method for virus removal) is the most expensive downstream step, accounting for up to 40% of costs. Thus, it is understandable that this step is a target for process optimization. Overall productivity, however, is not only a matter of initial flow rate, but also of total throughput and sensitivity to aggregate and impurity level. A viral clearance study with at least four viruses for late-stage development is state-of-the-art, and log reduction values (LRVs) of 4 and higher are hygienic factors. More interesting, but also less trivial, are LRVs that are independent of the flow decay of the filter.

Other Current Trends in Bioseparation

A clear trend in bioseparations is to combine steps, even eliminating the need for staging tanks and hold-up volumes between unit operations. Orthogonal strategies for each objective are key to achieving industry goals for yield and quality.

With increasing biomass in cell culture, high-productivity harvesting procedures are enticing, and there is a lot of interaction among the three unit operations of centrifugation, cross flow, and depth filtration. Eventually, we will see more integrated processes, such as expanded bed adsorption, ideally in disposable formats. For monoclonal antibody manufacturing, for example, we need to develop processes that require only two column steps followed by a rational filtration train, in which every step addresses the removal of a certain contaminant.

Polishing before capturing sounds provocative, but is becoming more common. This approach protects the protein A column and prevents the column eluate from precipitating by removing certain lipoproteins from the feed stream. These concepts can be custom designed with smart membranes that remove individual contaminants like proteases very specifically and early in the process to yield stable storage forms.

In chromatography, attempts are being made to make resins look more like membranes and vice versa. It makes much more sense to accept the limitations of both technologies. They each are great in their individual core application areas and deserve an integrated approach, for example, by using a chromatography train (capturing phase) followed by a filtration train (polishing phase). Capturing will always require high dynamic capacity and will be dominated by resins (with the exceptions of large molecules and high dilutions), whereas polishing is going to be the domain of charged membranes.

Thus, we are seeing many paradigm shifts in bioseparations, including the trend toward disposables to save costs. I predict that in a number of years, polishing applications will be dominated by disposable concepts. But much more is required. If downstream processing had kept pace with the revolution in fermentation, chromatographic separations would require only minutes, not hours. Because this is not feasible, we must seek alternatives. Bioseparation is desperate for high-throughput, high-productivity unit operations. One place to start would be to copy what has been done in food, beverage, and technical enzyme manufacturing.

In sum, robust and reliable methods such as extraction, precipitation, and crystallization deserve a renaissance. Michelangelo, although considered a master, was said to be constantly dissatisfied with himself. He probably realized that complacency is a dangerous enemy. We should take our cue from him.


1. BioPlan Associates. 3rd annual report & survey on biopharmaceutical manufacturing capacity and production. June 2005.

2. Wurm FM, Production of recombinant protein therapeutics in cultivated mammalian cells. Nature Biotechnol. 2004; 22: 1393–1398.

3. Aldridge S. Downstream processing needs a boost. GEN 2006; 26(1).

4. Zhou JX, Tressel T. Basic concepts in Q membrane chroma-tography for large scale antibody production. Biotechnol. Progress. 2006; 22(2):341–349.

5. Sinclair A. Disposable technologies—impact on biomanu-facturing. London: Institution of Chemical Engineers; 2005.

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