Preparative Separation of Proteins Using Centrifugal Precipitation Chromatography Based on Solubility in Ammonium Sulfate Solution

Abstract

A preparative modification of the centrifugal precipitation chromatography (CPC) is described. The sample-loading capacity is improved in the present system by the use of convoluted tubing containing dialysis tubing instead of a dialysis membrane placed between a pair of disks equipped with mirror-imaged spiral grooves as in the original design. The system uses, basically, the same principle of as the original CPC, in that a concentration gradient of precipitant is generated under a centrifugal force field. The protein sample injected into the CPC column is exposed to an increasing concentration of the precipitant where it precipitates at various portions of the column according to its solubility. The gradient is then gradually lowered so that the sample undergoes dissolution and precipitation many times within the column; the proteins finally elute from the column according to their solubilities. A basic study was performed using this machine to separate human albumin and 3-globulin using ammonium sulfate (AS) as precipitant. Preliminary results indicate that this method can separate 500 mg of protein.

Preparative Fractionation of Protein, RNA, and Plasmid DNA Using Centrifugal Precipitation Chromatography with Tubular Dialysis Membrane Inside a Convoluted Tubing as Separation Channel

“…The design of the preparative-scale CPC provides a much larger membrane surface area for mass transfer (21). As numerically shown in Table 2, the ratio between total surface area of the dialysis membrane tubing and total length of the separating channel in preparative-scale CPC is about 5 times larger than that of the bench-scale.…”

“…Higher flow rates allow a higher rate of precipitant migration through the membrane and thus better concentration gradient formation and may as well facilitate operation of the preparative-scale CPC. Centrifugal force applied to preparative-scale CPC is lower than that to the bench-scale unit, and the value is obtained from that in the routine operation of these two units set by their inventor (21).…”

Overcoming challenges of improving legacy biopharmaceutical processes

“…These techniques incorporate unit operations such as perfusion cell culture, continuous chromatography and single‐pass tangential flow filtration (TFF) 25–27 . ATPS have also been considered for use in continuous operation together with technologies such as mixer‐settler units, centrifugal partition chromatography and counter‐current chromatography, with the potential of integrating several upstream and downstream unit operations for product recovery, concentration and partial purification 28,29 . Continuous processing may improve process throughput and help to address product shortage issues, and it may be particularly attractive for biopharmaceutical products with high commercial demand.…”

Integration of aqueous (micellar) two-phase systems on the proteins separation

“…These methods are easy to validate and implement in batch and larger scale, however they are quite expensive. Among the non-chromatographic methods, ultrafiltration [7,8] and precipitation [9,10] appear as the main approaches used in protein separation, though, these methods are ineffective in the separation of similar proteins, since they only act based on protein size and hydrophilicity, respectively. Over the last years, aqueous two-phase systems (ATPS) emerged as an alternative platform for protein separation, considering their intrinsic versatility, in some cases leading to an enhanced purification performance [11].…”