Continuous Protein Purification Research Articles

Digital Integration of Laboratory Devices Using SiLA 2: Continuous Protein Purification Using Periodic Counter‐Current Chromatography

Digital Integration of Laboratory Devices Using SiLA 2: Continuous Protein Purification Using Periodic Counter‐Current Chromatography

Development of a Microfluidic Platform for R-Phycoerythrin Purification Using an Aqueous Micellar Two-Phase System.

Temperature-dependent aqueous micellar two-phase systems (AMTPSs) have recently been gaining attention in the isolation of high-added-value biomolecules from their natural sources. Despite their sustainability, aqueous two-phase systems, and particularly AMTPSs, have not been extensively applied in the industry, which might be changed by applying process integration and continuous manufacturing. Here, we report for the first time on an integrated microfluidic platform for fast and low-material-consuming development of continuous protein purification using an AMTPS. A system comprised of a microchannel incubated at high temperature, enabling instantaneous triggering of a two-phase system formation, and a microsettler, allowing complete phase separation at the outlets, is reported here. The separation of phycobiliproteins and particularly the purification of R-phycoerythrin from the contaminant proteins present in the aqueous crude extract obtained from fresh cells of Gracilaria gracilis were thereby achieved. The results from the developed microfluidic system revealed that the fractionation performance was maintained while reducing the processing time more than 20-fold when compared with the conventional lab-scale batch process. Furthermore, the integration of a miniaturized ultrafiltration module resulted in the complete removal of the surfactant from the bottom phase containing R-phycoerythrin, as well as in nearly twofold target protein concentration. The process setup successfully exploits the benefits of process intensification along with the integration of various downstream processes. Further transfer to a meso-scale integrated system would make such a system appropriate for the separation and purification of biomolecules with high commercial interest.

Accounting for interface behaviour in multi-stage aqueous two-phase extraction

Aqueous two-phase extraction (ATPE) is an alternative, cheaper and continuous protein purification technique. For ATPE to be used in industry, it must compete with current batch purification procedures, which can be achieved using multi-stage operation. To design extraction processes using ATPE appropriate process models must be developed. In this study, experimentally determined single-stage equilibrium data was used to generate a model to describe the behaviour of protein in multi-stage counter-current ATPE. Two distribution systems are considered: liquid–liquid (LL) and liquid-interface-liquid (LIL) distribution. The LIL model considers material which precipitates at the interface of the system. These models were then both compared against a three-stage experimental case study ATPE. The LIL model described the case study system better than the LL model, reducing the error from 40% to 11%. With this significant increase in accuracy, the LIL model represents an important tool with which to design multi-stage ATPE processes.

Single pass diafiltration integrated into a fully continuous mAb purification process.

The concept of continuous manufacturing has gained significant interest from the biopharmaceutical industry over the past several years. Benefits include increased manufacturing productivity, improved quality control, reduction in plant footprint, and more flexible management of facility capacity. There are several technologies currently available that enable continuous processing for chromatography and ultrafiltration. However, a single pass diafiltration design that meets the required small molecule clearance and has been integrated into a fully continuous monoclonal antibody purification process has not been previously published. Here, the theory and design of a 3-stage single pass diafiltration step is presented. Buffer exchange greater than 99.75% was experimentally demonstrated. Several critical design aspects were incorporated to minimize system complexity and reduce the buffer volume requirements. Lastly, single pass diafiltration was demonstrated in a pilot scale continuous process with uninterrupted flow from the bioreactor through the formulation step. This work illustrates the feasibility of incorporating a single pass diafiltration step into an end-to-end continuous protein purification process.

Single Pass Diafiltration Integrated into a Fully Continuous mAb Purification Process.

The concept of continuous manufacturing has gained significant interest from the biopharmaceutical industry over the past several years. Benefits include increased manufacturing productivity, improved quality control, reduction in plant footprint, and more flexible management of facility capacity. There are several technologies currently available that enable continuous processing for chromatography and ultrafiltration. However, a single pass diafiltration design that meets the required small molecule clearance and has been integrated into a fully continuous monoclonal antibody purification process has not been previously published. Here, the theory and design of a 3-stage single pass diafiltration step is presented. Buffer exchange greater than 99.75% was experimentally demonstrated. Several critical design aspects were incorporated to minimize system complexity and reduce the buffer volume requirements. Lastly, single pass diafiltration was demonstrated in a pilot scale continuous process with uninterrupted flow from the bioreactor through the formulation step. This work illustrates the feasibility of incorporating a single pass diafiltration step into an end-to-end continuous protein purification process. This article is protected by copyright. All rights reserved.

Effect of zinc chloride and PEG concentrations on the critical flux during tangential flow microfiltration of BSA precipitates.

There is renewed interest in the possibility of using precipitation for initial capture of high value therapeutic proteins as part of an integrated continuous downstream process. These precipitates can be continuously washed using tangential flow filtration, with long term operation achieved by operating the membrane modules below the critical filtrate flux for fouling. Our hypothesis was that the critical flux for the precipitated protein would be a function of the properties of the precipitate as determined by the precipitation conditions. We evaluated the critical flux using a flux-stepping procedure for model protein precipitates (bovine serum albumin) generated using a combination of a crosslinking agent (zinc chloride) and an excluded volume precipitant (polyethylene glycol [PEG]). The critical flux varied with shear rate to approximately the 1/3 power, consistent with predictions of the classical polarization model. The critical flux increased significantly with increasing zinc chloride concentration, going from 60 L/m2 /h for a 2 mM ZnCl2 solution to 200 L/m2 /h for an 8 mM ZnCl2 solution. In contrast, the critical flux achieved a maximum value at an intermediate PEG concentration. Independent measurements of the effective size and viscosity of the protein precipitates were used to obtain additional understanding of the effects of ZnCl2 and PEG on the precipitation and the critical flux. These results provide important insights into the development of effective tangential flow filtration systems for processing large quantities of precipitated protein as would be required for large scale continuous protein purification by precipitation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1561-1567, 2017.

Continuous protein purification using functionalized magnetic nanoparticles in aqueous micellar two-phase systems

A novel technique for technical-scale continuous purification of proteins is presented. It is based on the combined use of functionalized magnetic nano-particles and an Aqueous Micellar Two-Phase System featuring the non-ionic surfactant, Eumulgin ES, which undergoes temperature induced phase separation at ∼25°C. In the first step, conducted below the transition temperature (i.e. 15°C), the magnetic sorbent particles are added into the single dispersed phase and bind the protein of interest. Next, on raising the temperature to 30°C the protein-laden magnetic particles partition strongly into the micelle-rich top phase of the micellar two-phase system that's formed. The magnetically susceptible top phase is then continuously separated from the micelle-poor phase in a flowthrough tailor-made magnetic extractor featuring a permanent magnet providing an upwardly acting magnetic force. This separation device was shown to be effective for continuous separation of a wide range of differently sized magnetic particle sorbents (i.e. from 2μm diameter to as small as 25nm) from a 10% (w/w) Eumulgin ES system; high separation efficiencies were recorded for the phase-forming surfactant (87 to >98%), and all magnetic sorbent particles tested (95–99.9%). Finally, protein purification by continuous magnetic extraction was demonstrated at 15L scale for the recovery of an antibody fragment, A33 Fab′, from a crude extract of Escherichia coli periplasm. Nearly 70% of the A33 Fab′ initially present in the extract at 15.6% of the total protein content was recovered in a 2-fold concentrated and highly purified (>98%) state. Further, the amounts of magnetic sorbent and phase-forming surfactant lost in the process were very small; thus recycling of both components into subsequent rounds of continuous magnetic extraction is highly feasible.

Periodic counter‐current chromatography – design and operational considerations for integrated and continuous purification of proteins

Integrated and continuous processing of recombinant proteins offers several advantages over batch or semi-batch processing used traditionally in the biotechnology industry. This paper presents a theoretical and practical approach for designing a periodic counter-current chromatography (PCC) operation as a continuous capture purification step that is integrated with a perfusion cell culture process. The constraints for continuous and optimal PCC operation govern the selection of residence time and number of columns. The flexibility available in PCC design for selection of these parameters is dictated by the binding characteristics of the target protein on the capture resin. Using an empirical model for the protein breakthrough curve, analytical solutions to determine these conditions were derived and verified with experimental results for three different proteins: two relatively unstable proteins (recombinant enzymes) and a relatively stable protein (monoclonal antibody). The advantages of a continuous downstream capture step are highlighted for the three case studies in comparison with the existing batch chromatography processes. The use of PCC leads to improvements in process economics due to higher resin capacity utilization and correspondingly lower buffer consumption. Furthermore, integrated and continuous bioprocessing results in a smaller facility footprint by elimination of harvest hold vessels and clarification, as well as by reducing the capture column size by one to two orders of magnitude.

Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags

High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device.

Scale-down of continuous protein purification by annular chromatography: Design parameters for the smallest unit

Theoretically, in fixed-bed chromatography an infinite separation time or volume is available. In annular chromatography the separation time is limited by the time required for a single rotation. In a chromatograph with a smaller annulus than the theoretical one, the equilibration zone will overtake the feed zone. The required separation time is determined by a number of steps such as equilibration, loading, washing, elution, regeneration and the retention of the solute. A mathematical model has been developed to estimate the minimum radius of an annular chromatograph. The minimal geometry of an annular chromatograph was calculated for a model system immunoglobulin and bovine serum albumin.

Modeling of the continuous counter-current expanded bed adsorber for the purification of proteins

Mathematical models have been developed to describe the performance of continuous counter-current expanded bed adsorbers, novel devices for continuous purification of proteins from unclarified preparations of disrupted cells. Predictions from the models have been compared with experimental data from small scale systems. These models have been used to illustrate how the performance of the adsorber can be optimised by appropriate choices of contactor dimensions and other operation conditions. In addition, the work also illustrates how the device can be expected to deliver superior performance when compared to more conventional counter-currently operated systems, such as series of mixer-settler units.

A method for the continuous purification of proteins by affinity adsorption

A method was conceived for the purification of biomolecules on a continuous-flow basis using affinity adsorption. The affinity ligand was bound to a nylon belt which was passed sequentially through four chambers to which flows of feedstock, wash medium, eluent and regeneration medium were applied. The target compound was thus removed from the feedstock stream and desorbed into a continuous flow of eluent. Prototype laboratory-scale apparatus was designed and constructed and the technical feasibility of this method was demonstrated using soybean trypsin inhibitor as a ligand for the adsorption of trypsin. The effects of various operational parameters on apparatus function were investigated using this model system. In addition, continuous removal of trypsin from a bovine pancreatic extract was carried out during an 8 h experiment.

Novel affinity separations based on perfluorocarbon emulsions: Development of a perfluorocarbon emulsion reactor for continuous affinity separations and its application in the purification of human serum albumin from blood plasma

Perfluorocarbon affinity emulsions are generated by the homogenisation of a perfluorocarbon oil with a polymeric fluorosurfactant previously derivatised with an affinity ligand and subsequently cross-linked in situ. This procedure gives rise to a novel liquid affinity adsorbent that can be used for continuous protein purification. Discrete emulsion droplets were found to be unstable when pumped for prolonged periods; however, when flocculated, the emulsion floccules with diameters of around 125 μm, were very stable and sedimented faster. A four-stage reactor unit (perfluorocarbon emulsion reactor for continuous affinity separations, PERCAS) was designed and constructed to carry out continuous separations, and exploited the unusual properties of the adsorbent, i.e. liquid nature and high density. Each of the four stages of PERCAS consisted of a mixing tank, for contacting between emulsion phase and aqueous phase, adjacent to a settling tank for the subsequent separation of emulsion from the aqueous phase. Using PERCAS adsorption, washing, elution and re-equilibration of the emulsion could be carried out continuously with emulsion recycle. Using single-component adsorption of human serum albumin to a perfluorocarbon affinity emulsion derivatised with the triazine dye C.I. Reactive Blue 2, PERCAS was optimised with respect to flow-rates and input concentrations. The work was then extended to the continuous purification of essentially homogeneous human serum albumin from blood plasma.

Novel affinity separations based on perfluorocarbon emulsions: Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000–10 000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 ± 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.

Mathematical modelling of the continuous affinity-recycle extraction purification technique

Continuous affinity-recycle extraction (CARE), a continuous protein purification unit operation, has been designed to address design and optimization criteria relevant for process scale chromatographic separation of proteins. The development and application of a mathematical model describing purification in the CARE process are described. The model incorporates adsorption-desorption kinetics into material balance equations describing the operation of two well-mixed reactors operating with recycle. An accurate mathematical model of CARE has aided in its development as a new unit operation for protein purification, in the assessment of its performance tradeoffs, and in its optimization.