Throughout the ages, great sculptors like Michelangelo have said that their work is about taking a block of marble and freeing a form that is already there. All the artist needs to do is remove everything that does not belong in the masterpiece.
In reality, there is a conflict between upstream and downstream processes resulting from the industry's strength in fermentation and weakness in purification. The consequences are dramatic. Monoclonal antibodies (MAbs)—the fastest growing biopharmaceutical market segment, with predicted annual sales of $20 billion by 2010—are an example. Demand has already reached 6 metric tons this year.Fortunately, large amounts of raw material are accessible as a result of sufficient manufacturing capacity that has become available recently, combined with significant productivity increases in upstream processes.1 Experts claim that even after recent dramatic improvements from mg/L to g/L of antibody titers in mammalian cell culture, the best is yet to come.2 From the early days of MAb manufacturing, we know that physiological levels of up to 20 g/L can be achieved in mouse ascites. That is probably the long-term goal we are looking at.
Unfortunately, we have not seen the same speed of progress further downstream. Downstream operations have always been in a sandwich position. Typically, senior management focuses on the initial phase of the process, where most investment goes and where the raw material is generated. The other typical focus is formulation, as the outlet for the drug product. For years, downstream simply had to deliver; nobody asked how. Now, as the bottleneck becomes obvious, there is a lot of pressure on purification. We are in a technology crisis, and protein purification must make a comeback.3
Today's downstream processing operations generally focus on two main areas: a) the initial recovery phase, including capturing, where bulk purity is achieved; and b) the subsequent polishing phase, which adds safety through orthogonal strategies for impurity and pathogen clearance.
It is relatively easy to identify the bottlenecks in the process. Early recovery, including harvesting, cell removal, and clarification, is not a problem. These steps can be operated with robust technology that processes high volumes in the biopharmaceutical industry; and even larger volumes—beyond the 100,000-L scale—in food, beverage, and technical enzyme manufacturing.
But further downstream, for example in capturing, processes are dominated by chromatography with its inherent limitations. Very large scale chromatography is technically feasible, but big columns encounter big problems and current upstream titer developments are pushing biochromatography beyond its physical and economic limits.
Protein A is the work horse in antibody separation, and accounts for up to 10% of the overall costs; individual column fillings cost millions of US dollars but still cannot cope with the volume of common fermenter batches. Despite such problems, however, Protein A offers unmet specificity, so it will continue to be the industry standard until a breakthrough arises from the second or third generations of the mimetic ligands that are currently available.
Because state-of-the-art capturing, including affinity chromatography, yields material that is >98% pure, polishing efforts focus on removing pathogens and trace contaminants. Because this area does not receive much attention, polishing does not seem to represent a bottleneck. In reality, it does, because in the standard approach— removing typical process-derived contaminants with anion exchangers in flow-through mode—the flow rate, rather than binding capacity, becomes the limiting factor. Thus, we run into a problem: to increase the flow rate, we need very large chromatography columns, which require very large quantities of expensive resins.