In the less than 20 years since Marcel Raedts founded PROXCYS Downstream Biosystems B.V. in 2003, it has specialized in producing high-performance, low-pressure radial flow chromatography (HP-RFC) columns and systems. Applications of the HP-RFC columns and systems include purifying biologicals from a vareity of sources, including blood plasma, cell cultures and bacterial fermentation. 

Radial flow chromatography and traditional chromatography columns have key differences, according to Thijs Groenewegen, technical sales engineer for PROXCYS. “Traditional columns flow from top to bottom, and while the RFC separation principle is the same, the column design is different,” said Groenewegen, in the company’s marketing materials. “Instead of top to bottom, the RFC flow is from the outside to the inside of the column.” 

Turning the cylinder on its side results in a smaller footprint.

While traditional columns are common in the marketplace, the marketshare for HP-RFC columns has grown over the past decade plus. Already popular when used with human plasma products, the demand for pre-packed SUPR RFC columns for cell culture/fermentation-biopharmaceuticals is also expanding swiftly. 

Overcoming chromatography hurdles

A standard purification step in bioprocessing, chromatography involves separating components in a mixture. 

The step can be used to separate and purify the biopharmaceutical drug or target molecule from other molecules and impurities in the mixture. This purification process takes place in a chromatography column, a cylindrical vessel fully packed with 50–100 µm chromatography beads, also called resin or sorbent, immobilized inside the column. 

In the purification process, the mobile phase — the mixture of product and impurities — enters the column and passes the resin (stationary phase). 

Selective binding, or adsorption, of the target molecules to the chromatography beads causes the separation. 

The adsorption can be influenced by a number of operating conditions, including conductivity, pH, salt type and other operating conditions. 

Traditional chromatography-column design is “axial.” The chromatographic media are packed in a relatively flat, broad, pancake-like cylinder in this setup. The liquid stream passes through it vertically, from top to bottom, or vice versa. 

If the goal is to increase the manufacturing capacity of a chromatography column, increasing the column height is limited as a result of the increase of back pressure within the system. 

This limitation means increasing the column diameter is the only viable option to increase the scale. The result is a system with a short, wide bed that is not efficient in terms of the space required and its overall weight. In addition, there is also the growing difficulty in consistently creating a high-performing packed bed. Therefore, in axial chromatography, packed beds are typically taller than necessary to reduce the diameter of the axial columns, with an accepted tradeoff: reduced throughput but increased operating pressure. 

On the other hand, RFC, provides high throughput and reduced pressure drop. The system’s weight and footprint can be one-quarter of that of axial-flow columns. Unlike axial-flow systems where the liquid stream passes through the chromatography media vertically, RFC sees the media wrapped around a central cylinder. The liquid stream passes through it horizontally by applying positive pressure. RFC essentially folds the horizontal bed of the axial system into a vertical cylinder and changes the flow direction from vertical to horizontal. While the purification and fractionation capabilities remain of the process remain the same, the system’s footprint and weight are radically reduced. 

Therefore, the key to optimized performance in an HP-RFC column is proper packing of the chromatographic media – usually some high-value resin – in the tube through which the liquid stream passes. 

“Proper column packing has a direct result on the performance of the column,” said Groenewegen. “A well-packed column contributes directly to the separation and efficient capture of the intended product. So, the better the resins are packed, the better the separation and the better the resolution of the targeted proteins.” Properly packing a column, however, is often easier said than done.

Packing traditional columns is getting complicated when the ratio of the column diameter over the bed height is over 8 to 10 inches. With HP-RFC, you can easily go beyond 30 inches, which means the bed height can easily be reduced (lower pressure), or the column volume can be huge. Additionally, HP-RFC columns are always pump-packed, which facilitates the automation of the system, and with the proper equipment, even the extreme formats can be packed effortlessly. 

“To develop a perfect packing, a lot of variables have to be taken into account – flow speed, back pressure, slurry concentration and resin quantity,” said Groenewegen. “The characteristics of the resin beads themselves are also different; some resins are quite fragile and need to be handled gently, while some are quite rigid and can be moved with more force. However, when the parameters are known or optimized, HP-RFC packing is extremely simple, reproducible and reliable.” 

Picking the proper pump 

Critical to proper packing of any resins into the HP-RFC column is using the proper pumping technology. For the past decade, PROXCYS has incorporated four-piston diaphragm pump technology into its proprietary column-packing systems. 

“The pumps are accurate, have a large turndown ratio, have a compact design for easy integration into our small systems, they don’t create foam when they’re pumping, and their action is ‘fast but gentle’ to allow shear-sensitive resin beads to be pumped without damage,” said Groenewegen. “(These) pumps are excellent for projects where you need a gentle pump, but one that still produces high flow rates and is easy to control.” 

The main advantage of the use of four-piston diaphragm pumps in HP-RFC applications is its unique form of operation: The four diaphragms are driven one after another by a connector plate, which moves back and forth out of its central position in a stroke that is generated by an eccentric shaft, with the length of the stroke determined by the angle of the eccentricity. In other words, the technology has been modeled on the operation of the human heart – which is eminently capable of pumping whole human blood, one of the most shear-sensitive products in the world – with its four pumping chambers and check valves keeping product flow constantly moving forward. 

The pump chambers also contain no rotating parts that can be subject to friction, which minimizes any operational heat buildup that can compromise the product.

This mode of operation also means that the pumps can run dry, self-priming, and create low shear because of low slip. In addition, they offer low-pulsation, leak-free operation while having great dry/wet suction-lift capabilities and are easy to control to achieve the required flow rates or pressures. In other words, they possess all of the required characteristics for proper RFC column packing. 

At one point, PROXCYS tried using a different pump technology. Unfortunately, these pumps – which had two rather than four diaphragms – underperformed and had problems.

“After a few minutes, the pump stopped because the resin beads were stuck between the membranes of the pump,” Groenewegen said. 

In recent years, the bioprocessing market has seen increased demand for pumps with replaceable heads, which can be achieved by using single-use versions of this pump technology. This is attractive to the growing number of manufacturers who wish to use pumps with heads that can be simply changed out after production runs rather than installing a permanent stainless-steel production line that will need to be cleaned after each production run. 

“If you have a batch process, they like to swap the single-use head with a new one and then move on to the next campaign,” Groenewegen said. “Personally, I try to push the single-use pumps because I think that’s the direction the industry is moving toward, and it is also pairing with our own strategy promoting the SUPR pre-packed HP-RFC column line.”