Properties of hydrodynamic J-type countercurrent chromatography for protein separation using aqueous two-phase systems: With special reference to constructing conical columns

Highlights

• The conical column is constructed on J-type countercurrent chromatography (CCC).

• The CCC conical column shows decent stationary phase retention.

• The CCC conical column shows sound phase mixing.

• Experimental conical columns were constructed based on prior theoretical analysis.

• Proteins are successfully separated using constructed real conical columns.

Abstract

Protein separation using hydrodynamic countercurrent chromatography (CCC), where low backpressure is inherent, is more challenging, more time consuming and more costly when compared with separating small molecules. The most hopeful approach is to rationally design suitable columns for already commercialized J-type CCC machinery. By comparing 3 column geometries (3D helix, 2D spiral and 3D cone), we firstly constructed the mechanical model tailored to the conical column on J-type CCC using aqueous two-phase system (ATPS) on protein separation. Aimed at mechanistically understanding hydrodynamic CCC, we then developed a semi-quantitative model to account for contributions of both hydrodynamic and hydrostatic forces to stationary phase retention, and have subsequently compared the modelling outcomes with experimental results. We practiced a methodology to delineate both phase mixing and stationary phase retention before committing to physically constructing CCC columns. Following theoretical analyses, we finally constructed conical columns for J-type CCC. Using model proteins (myoglobin and lysozyme) and with 2 ATPSs containing PEG1000 and phosphate, sound protein separation has been achieved (resolution reaches 1.5-2.0 and stationary phase retention also exceeds 40%) for the selected ATPSs and under a varied level of sample volumes and loadings.

Introduction

As an alternative technology to liquid-solid chromatography, high-speed countercurrent chromatography (CCC)has gained a market share in preparatory/industrial separation and purification on bioactive chemical components from natural resources [1], [2], [3]. When applied to proteins and the large molecules alike, this technology has met with persistent challenges, which are particularly daunting if our aim has been orientated towards large-scale manufacturing usages rather than analytical needs. CCC is a dynamically cyclic process, and there is only limited times for phase mixing and phase separation in each cycle, both of which are essential for partition chromatography like CCC. Targeted and impurity small molecules can reach thermodynamic equilibration under only a low mixing intensity for e.g. 3D helical column J-type CCC. For large molecules, thermodynamic equilibration cannot be reached within times allowed unless under a high phase mixing intensity. Consequently, there are 2 big issues that are uniquely associated with large molecules and nano-particles: (a) how to overcome technology barriers for achieving thorough phase mixing and, (b) how to overcome difficulties in achieving satisfactory stationary phase (SP) retention. The first issue results from sizes of the targeted molecules and/or particles, and the second from properties of 2 phase systems, typically aqueous two-phase systems (ATPS) required in such applications. Per the CCC working mechanism, separation of targeted molecules relies on differentiation in their partition coefficients in a 2-phase system. The role of thermodynamics in driving protein partition has been considerably dwindled by increased molecular dimension, and consequently realization of its CCC separation resolution potential depends greatly on phase mixing intensiveness. In the absence of net hydrostatic force − largely centrifugal force − in tangential directions of the CCC column, similarity in hydrophilicity between the 2 phases is invariably detrimental for reaching a decent SP retention [4], [5]. Unlike CCC, the flow channel of CPC is not a continuous tubing and so phase mixing is often caused by suddenly narrowing down sectional areas of mobile phase (MP) flows [6]. Such feature of CPC makes it challenging to comply with quality by design GMP requirement definitively needed in biopharmaceutical industry [7].

The conventional definitions on CCC and CPC have been based on defined devices where CCC undergoes a planetary motion and CPC only circular motion. Correspondingly, conventional CCC has been termed as hydrodynamic CCC, and CPC as hydrostatic CCC [8]. Paradoxically, toroidal column J-type CCC is more hydrostatic than hydrodynamic [9]. By all means, such device-based definition has narrowed our view in understanding and then developing the potential of CCC technologies.

Unlike the strategy for separating small molecules where solvent system selection plays a pivotal role, separation of large molecules relies critically on the column geometry for hydrodynamic CCC [10]. A prominent problem for hydrostatic CCC is invariably high backpressure, which eventually imposes increased manufacturing costs on the targeted products. Alternative to ATPSs yet using J-type CCC, reverse micelle (RM) systems have been encouragingly explored for separating model proteins [11], bromelain [12], and another proteinase [13]. Nevertheless, the phase-forming components for RM systems are invariably toxic and presently it bears no hope for using them in biopharmaceutical processing.

For hydrodynamic CCC, the best solution discovered so far for improving SP retention has been to adopt 2D spiral columns [14], [15], [16], [17], [18], [19]. Such column works by obtaining, in a column tangential direction, a small proportion of the hydrostatic force resulted from the CCC planetary motion [20]. Ito developed two spiral column assemblies: the spiral disk and the spiral tubing. Phase mixing in spiral disks for protein separation is achieved using glass beads in each barricaded chamber. To improve phase mixing in spiral tubing, the plastic tubing sectional geometry has been modified in various ways [16], [21], [22], [23], [24]. Only the 8-shaped column has been shown to have potential for protein separation at an incredibly slow MP flow rate [21].

To greatly improve the subsequent trial-and-error efficiency, we developed the present quantitative approach, which is based on classic mechanics, as a pre-experimental screening on J-type CCC column geometries. Broadly speaking, there are 2 modelling regimes in quantitatively describing dynamic events taking place inside CCC columns. The first regime is analogy modelling where the modeler assumes that thorough phase mixing and ideal phase separation take place alternatively along the longitudinal length of a CCC column [25], [26], [27], [28], [29]. Under such assumption, use of countercurrent distribution (CCD) to simulate CCC can not only designate a chromatographic peak retention time but also present an entire peak [25]. This approach may be used to confirm if the performance of an existing column, but cannot provide a priori info as to what column geometry would work prior to physical construction. The second regime is mechanical modelling where the modeler follows the featured CCC planetary motion, case by case, to describe dynamic changes of all primary forces at all column locations [20], [30], [31]. This type of models is appropriate for the a priori analysis on both phase mixing and phase retention. Good agreements between experimental observation and modelling analyses had been obtained for 2D spiral column [20], [30] and 3D helical column [31].

For protein separation and purification, a new type of CCC column, conical-shaped column, has been proposed in this work. Although having been used for separating small molecules [32], [33], this CCC type has not been indicated and justified for separating proteins or the other large hydrophilic molecules. Prior to physically constructing such columns, the first objective of the present work was to establish tailored mechanical model for conical columns on J-type CCC and then to assess its suitability for protein separation. Geometrically, 3D conical has similarities to both 2D spiral and 3D helix, and conceivably J-type CCC with these column geometries are all hydrodynamic. Based on already observed experimental results mainly on stationary phase (SP) retention, the second objective was to examine if there are common properties to any hydrodynamic CCC. Based upon modelling outcomes, the third objective was to physically construct modelled conical columns and then conduct experiments on separating model proteins.