A z2 laterally-fed membrane chromatography device for fast high-resolution purification of biopharmaceuticals
Introduction
Membrane chromatography (MC) is a rapid and scalable alternative to conventional resin based column chromatography [1], [2], [3], [4], [5], [6]. As solute binding takes place on the walls of flow-through pores within the membrane, the predominant mode of solute mass transport in MC is convection [1], [2], [3], [4], [5], [6]. The large frontal area in MC devices allow these to be operated at low pressures. Therefore, MC is more than an order of magnitude faster than resin based column chromatography [3]. The predominance of convection also makes separation by MC relatively less sensitive to the flow rate [3,4,7,[8], [9], [10]. Another significant advantage of MC is its suitability for single-use applications [11], [12], [13], [14], which eliminates the need for time-consuming and labour-intensive steps such as cleaning and validation [4,5,7,10,15]. Unlike a resin-packed column, whose performance is critically dependant on packing skills [16], membrane chromatography devices of consistent quality can easily be mass produced [15,17,18]. However, one of the major limiting factors with conventional MC devices such as the stacked disc and the radial flow devices is poor resolution [19], [20], [21]. For instance, it has been shown that a radial flow MC device cannot be used for carrying out high-resolution, multicomponent bind-and-elute separations [20]. This limitation could be attributed to the non-uniform flow within the device [19], [20], [21]. Resolution in MC could potentially be enhanced through the use of improved devices such as the laterally-fed membrane chromatography (or LFMC) device [19], [20], [21], [22], [23], [24], [25], [26]. A head-to-head comparison study between an LFMC device and its equivalent commercial radial flow MC device showed that while the latter was totally unsuitable for carrying out high-resolution bind-and-elute separation, multi-component mixtures could be very efficiently fractionated using the equivalent LFMC device [20]. Another study showed that even in the flow-through mode, an LFMC device outperformed its equivalent radial flow MC device [21].
An LFMC device relies on a tapered inlet for flow distribution, and a similarly tapered outlet for flow collection. While this worked well in the devices tested in our earlier studies, we recognize that a tapered inlet and outlet combination could potentially become a limiting factor while scaling up LFMC devices. Also, the separation performance of the LFMC device could potentially be enhanced further through design improvements. In a recent paper [27], we have described a novel z2 flow distribution and collection feature for improving the efficiency, compactness and scalability of a resin-based cuboid packed-bed chromatography device. The overall flow path within the z2 cuboid device could be visualized as a combination of two z-patterns. This z2 feature eliminated the need for a tapered inlet and outlet in the cuboid packed-bed device, led to significant improvement in uniformity of flow, and resulted in improvement in separation efficiency [27]. In our current study, we examine if the separation efficiency of an LFMC device could be enhanced by incorporating the z2 flow distribution and collection feature. We also describe a method for fabricating such a device.
The new LFMC device discussed in this paper is designated z2LFMC. Flow distribution and collection was achieved using primary and secondary flow channels as shown in Fig. 1A. The flow of fluid within the z2LFMC device could be classified at three levels of hierarchy, i.e. primary flow in the primary channels, secondary flow in secondary channels and tertiary (or normal) flow within the membrane stack. This three-level hierarchy was sustained by maintaining the hydraulic resistance in the membrane stack significantly higher than that in the secondary channels, which in turn was higher than that in the primary channels [27]. The arrangement of the flow channels in the device ensured that all hypothetical flow paths were of identical length. Also, the net hydraulic resistance along any hypothetical pathway was identical [27]. Hence, the residence time along any of these pathways could be expected to be the same. The use of straight flow channels in the z2LFMC device would also minimize back mixing. Therefore, the separation efficiency of an LFMC device could potentially be enhanced by adding the z2 flow distribution and collection feature. Also, the uniformity in path length in a z2LFMC device would be independent of the dimensions of the device. Therefore, such a device is expected to be highly scalable. Due to the asymmetric manner in which liquid is introduced to and removed from a z2LFMC device, a tracer solute front would tilt with respect to the directions of fluid flow along the x and z axes. Fig. 1B and C show the shape of the expected solute fronts along the x-y and y-z planes respectively, within a membrane stack housed in a z2LFMC device. This combination of slants is likely to help collimate the solute front at the outlet, thereby contributing towards the narrowing of the solute residence time distribution within the device [27]. z2LFMC devices having two different bed volumes (i.e. 5 and 15 mL) were fabricated using two different types of membranes. The 5 mL device housed a stack of strong anion exchange (Q) membrane with 0.8-micron pore size, while the 15 mL device housed a stack of strong cation exchange (S) membrane having pore size in the 3–5 µm range. These devices were first characterized in terms of their number of theoretical plates. The performance of a z2LFMC device was compared with an equivalent LFMC device having tapered inlet and outlet using computational fluid dynamic (CFD) simulations. The protein separation performance of the 5 mL Q z2LFMC device was compared with a 5 mL QFF resin based column, while the performance of the 15 mL S z2LFMC device was compared with a 15 mL Capto S ImpAct resin-packed column. The 15 mL S z2LFMC device was then used for a couple of biopharmaceutical purification case studies, i.e. the fractionation of monoclonal antibody charge variants, and the separation of monoclonal antibody aggregates. The results obtained are discussed.
Section snippets
Materials and methods
Mustang Q strong anion-exchange membrane sheets (MSTGQ3R) were purchased from Pall Canada Ltd. (Mississauga, ON, Canada). Sartobind S strong cation-exchange membrane sheets (94IEXS42–001) were purchased from Sartorius-Stedium Biotech (Gottingen, Germany). HiTrap QFF column (5 mL, 17–5156–01) and Capto S ImpAct (17–3717–01) resin was purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA). Millipore Vantage® L Laboratory Column VL 16 × 250 was kindly donated by PlantForm Corporation,
Efficiency assessment
In order to assess the impact of adding the z2 flow distribution and collection feature in the LFMC device, theoretical plate measurement experiments were carried out using sodium chloride as tracer solute. In these experiments, which were carried out at different flow rates, 0.4 M sodium chloride solution was used as the mobile phase while 0.8 M sodium chloride solution was used as the tracer solution. The volume of tracer solution injected to obtain the salt peaks was 1% of the respective
Conclusion
The separation efficiency of laterally-fed membrane chromatography could be enhanced by the incorporation of the z2 flow distribution and collection feature. The flow of fluid within a z2LFMC device could be classified at three levels of hierarchy. This was sustained by maintaining three levels of hydraulic resistances within the device, i.e. highest in the membrane stack, lower in the secondary channels and lowest in the primary channels. Experimental results showed that the number of
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
CRediT authorship contribution statement
Raja Ghosh: Conceptualization, Design of equipment, Experiments, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing - original draft, Writing - review & editing. Guoqiang Chen: Validation. Roxana Roshankhah: Validation. Umatheny Umatheva: Validation. Paul Gatt: Resource.
Acknowledgement
This work is supported by the Natural Science and Engineering Research Council (NSERC) of Canada. We thank PlantForm Corporation, Guelph, ON for donating the Millipore Vantage® L Laboratory Column VL 16 × 250, used in this study. We thank Prof. Geoff Hale, Dr. Pru Bird and other members of the Therapeutic Antibody Centre, Oxford University, UK for donating the hIgG1-CD4 and Campath-1H monoclonal antibodies examined in this study.
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2023, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life SciencesCitation Excerpt :Hence, the inlet and outlet for the device were located along the space diagonal of the rectangular membrane stack [27–29]. These plates were prepared by 3D printing [27], and the stack of strong cation exchange (S) membrane (20 mm width, 70 mm length, 10.7 mm height, and 15 mL membrane bed volume) was sandwiched and sealed in place between the two plates using a frame of polyurethane elastomer [26–28]. This device will be referred to in this paper as the 15 mL z2LFMC device.