These features have immediate practical value for industrial applications. One of the handicaps in downstream processing of antibodies has been the long residence time required to achieve high binding capacity with protein A affinity on porous particle media. The need for slow flow rates has often been attributed to kinetic limitations imposed by a low-affinity constant for IgG, but experimental data indicates that inefficient mass transport is the primary obstacle; binding reaches equilibrium 10 times faster on monolithic protein A. Dynamic binding capacity of DNA favors monoliths by 30- to 50-fold. Dynamic capacities in excess of 40 mg recombinant IgM/mL have been demonstrated on monolithic ion exchangers at flow rates of 15 column volumes per minute. This corresponds to a linear flow rate of 4,500 cm/hr on a 5 cm high bed of porous particles with the same volume.
All of these data speak to the potential for convective chromatography media, including membranes, to break the much-discussed bottleneck in downstream processing, the importance of which was highlighted in a presentation by Uwe Gottschalk, Ph.D.,vp of purification technologies from Sartorius Stedim Biotech. A presentation by Validated Biosystems indicated that although the dynamic capacity of current protein A monoliths is only about 10–12 g/L, continuous processing on a single 8 L radial flow monolith could purify 20 kg of IgG from 20,0000 L of cell culture supernatant in 27 hours, three times faster than a 19 L column of conventional protein A media with a capacity of 35 g/L.
Maarten Pennings, group manager at Tarpon Biosystems, presented another model projecting the ability of a 10 liter simulated moving bed (BioSMB™ ) array of 800 mL protein A monoliths to process 75 kg of IgG in 72 hours, the same time as a conventional column with a volume of 90 liters. The efficiency of BioSMB would enable the monolith system to achieve this result with buffer consumption equivalent to the conventional system despite its lower capacity. A BioSMB array of 8 L monoliths could process 75 kg in less than eight hours.
Both presentations concluded that the large number of process cycles in these models make it economical to dispose of the used monoliths after processing a single lot of IgG, thereby avoiding the need to develop and validate sanitization procedures.
Yow-Pin Lim, M.D., Ph.D., president and CSO of ProThera Biologics, discussed a monolith-based purification of Inter-alpha trypsin inhibitor, a 250 Kd plasma protein reported to be a highly effective treatment for sepsis and anthrax. He remarked on the ability of monoliths to accelerate process development, enabling more experiments in one day than can be completed with conventional media in a week.
“The speed of monoliths encouraged us to evaluate a much broader range of conditions than is typical during early-phase process development. The result was an ultrahigh efficiency anion exchange process that achieves greater than 90% purity and 75% recovery from plasma in a single step,” he said.
Another important difference between monoliths and particles concerns the void volume in particle columns. About 40% of a well-packed column is void space. Buffer follows the path of least resistance, flowing preferentially between the particles, and disfavoring solute contact with the particle surface, thereby depressing capacity.
An additional consequence of void space is the formation of eddies; vortexes created by differential friction between particle surfaces and the deeper void space. Eddies cause interstitial mixing that erodes resolution as solute zones flow down the column, and they create shear forces that may damage labile molecules. Eddy dispersion remains constant at higher flow rates, but shear increases in direct proportion.
Monoliths have no void volume; flow is laminar and they do not develop eddies.
The benefits of low-shear separations were highlighted in several presentations. Fatima Plieva, Ph.D., R&D scientist at Protista Biotechnology demonstrated passage of whole blood through a monolith with 10–200 µm channels without hemolysis. Similar monoliths have been used to capture von Willebrand Factor directly from whole blood, to capture inclusion bodies from high-debris lysates, and to capture live yeast and bacteria.
Kornelia Schriebl, Ph.D., research scientist at the Bioprocessing Technology Institute in Singapore, presented on stem cell purification and also remarked on the importance of low-shear chromatography supports for maintaining viability. In his keynote presentation, Charles Lutsch, Ph.D., director of downstream process development at Sanofi Pasteur, emphasized the importance of mild handling and rapid processing times for the manufacture of live and attenuated viruses and noted the ability of convective chromatography media to support both.