Dry-compression packing of hydroxyapatite nanoparticles within a flat cuboid chromatography device and its use for fast protein separation.

We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out at quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously increased by increasing flow rate. The suitability of this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.

A cuboid chromatography device having short bed-height gives better protein separation at a significantly lower pressure drop than a taller column having the same bed-volume.

Simultaneously reducing the bed-height and increasing the area of cross-section, while keeping the bed-volume the same, would substantially reduce the pressure drop across a process chromatography column. This would minimize problems such as resin compaction and non-uniformity in column packing, which are commonly faced when using soft chromatographic media. However, the increase in macroscale convective dispersion due to the increase in column diameter, and the resultant loss in resolution would far outweigh any potential benefit. Cuboid-packed bed devices have lower macroscale convective dispersion compared to their equivalent cylindrical columns. In this paper, we discuss how and why a flat cuboid chromatography device having a short bed-height gives better protein separation, at a significantly lower pressure drop, than a taller column having the same bed-volume. First, we explored this option based on computational fluid dynamic (CFD) simulations. Depending on the flow rate, the pressure drop across the flat cuboid device was lower than that in the tall column by a factor of 6.35 to 6.4 (i.e. less than 1/6th the pressure). The CFD results also confirmed that the macroscale convective dispersion within the flat cuboid device was significantly lower. Head-to-head separation experiments using a 1 mL flat cuboid device having a bed-height of 10 mm, and a 1 mL tall column having a bed-height of 25.8 mm, both packed with the same chromatographic media, were carried out. The number of theoretical plates per unit bed-height was on an average, around 2.5 time times greater with the flat cuboid device, while the total number of theoretical plates in the two devices were comparable. At any given superficial velocity, the height equivalent of a theoretical plate in the tall column was on an average, higher by a factor 2.5. Binary protein separation experiments showed that at any given flow rate, the resolution obtained using the flat cuboid device was significantly higher than that obtained with the tall column. This work opens up the possibility of designing and developing short bed-height chromatography devices for carrying out high-resolution biopharmaceutical purifications, at very low pressures.

High-resolution purification of a therapeutic PEGylated protein using a cuboid packed-bed device.

PEGylated proteins which are a class of protein-synthetic polymer conjugates that have shown significant promise in the area of biotherapeutics are difficult to purify. A cuboid packed-bed device was used to purify a mono-PEGylated therapeutic protein from impurities such as high molecular weight (HMW) species (e.g., tri- and/or di-PEGylated forms), and low molecular weight (LMW) species such as unreacted protein and polyethylene glycol (or PEG). The separation efficiency of this device was compared with that of an equivalent cylindrical column. The effects of operating conditions such as flow rate, buffer composition, elution gradient, and column loading were systematically compared. An equivalent column with the same bed volume, same resin and same bed height was served as control. In mono-PEGylated protein purifications experiments, the cuboid packed-bed device exhibited sharper peaks and gave better resolution at all conditions examined in this study. The purity of mono-PEGylated protein in the samples collected from the cuboid packed-bed device and the column were comparable, i.e., 98.1% and 97.9% respectively. The recovery of mono-PEGylated protein in the pooled eluate from the cuboid packed-bed device was 31.7% greater than that recovered in the pooled eluate from the column. Therefore, significantly higher recovery of mono-PEGylated protein was obtained with the cuboid packed-bed device while maintaining the same purity specification as obtained with the column.

A flow distribution and collection feature for ensuring scalable uniform flow in a chromatography device.

We discuss how the efficiency of a chromatography device could be enhanced by incorporating a new feature which ensures flow uniformity. The overall flow of fluid within the device, which has a cuboid shape, could be visualized as a combination of two z patterns. The device is therefore designated as cuboid z2. The reason for flow uniformity is explained using a simple mathematical model, and also based on computational fluid dynamic (CFD) simulations. The cuboid z2 device outperformed its equivalent conventional cylindrical column in terms of separation efficiency metrics such as the number of theoretical plates, the reduced plate height, and attributes of non-interacting tracer solute peaks. The results discussed in this paper clearly indicate the suitability of the cuboid z2 device for carrying out high-resolution chromatographic separations. The z2 flow enhancing feature would also be broadly applicable to any process which requires uniform flow in three-dimensional porous media. In addition to superior fluidic attributes, the cuboid z2 chromatography device has other advantages such as compactness and scalability.

Simulation and experimental study of the transport of protein bands through cuboid packed-bed devices during chromatographic separations.

Recent studies have demonstrated that box-shaped or cuboid packed-bed chromatographic devices represent an efficient alternative to conventional cylindrical columns for high-resolution preparative protein separations. This has been attributed to the greater uniformity of flow within these devices. However, for a more complete explanation, it is important to understand how the system hydrodynamics affects band broadening during the transport of proteins through these devices. In this study, we present first principle mathematical models to capture this interplay. These models were validated by flow-through and bind-and-elute experiments carried out using a colored protein as tracer. Control experiments were also carried out using equivalent commercial columns, i.e. having same bed height and cross-sectional area, and packed with same media. The trends observed in the experiments matched those predicted by the models, though there were deviations in the absolute values. These deviations are explained in terms of non-idealities that exist in the experimental set-up, as well as in terms of factors that were not considered in the model. The models discussed in this paper are not only useful for understanding the workings of the cuboid packed-bed device, but are also useful tools for designing, optimizing and scaling-up such devices.

Efficient capture of monoclonal antibody from cell culture supernatant using protein A media contained in a cuboid packed-bed device.

A box-shaped or cuboid packed-bed device was used for monoclonal antibody (mAb) separation using protein A affinity chromatography. The separation efficiency of the device was compared with an equivalent column i.e. packed with same resin, and having identical bed height and bed volume. The protein A media packed cuboid device had a larger number of theoretical plates than its equivalent column, e.g. 8750/m as opposed to about 4700/m at a flow rate of 0.5 mL/min. In mAb purification experiments, the impurity flow-through and eluted mAb peaks were shaper with the cuboid device. This implied that the effective separation time and buffer consumption with this device was lower, the purified mAb pooled volume was smaller, and the mAb concentration in the pooled volume was greater. Equivalent separation efficiency could be obtained with the cuboid device using higher flow rates than that used with the column. For instance, elution peaks equivalent to those obtainable by the column could be obtained at a 5 times greater flow rate using the cuboid device. The results discussed in this paper clearly demonstrate the potential for improving the efficiency of protein A affinity chromatography based mAb purification by using a cuboid packed-bed device.

Fast, low-pressure chromatographic separation of proteins using hydroxyapatite nanoparticles.

Columns packed with ultrafine particles (e.g. sub-2 µm porous particles) are suitable for high-resolution, high-speed analytical separation of proteins. However, they require very expensive chromatography systems to provide the ultra-high pressure required for carrying out separations using such columns. Also, frictional heating at high pressure could result in peak broadening and on-column protein degradation. In this paper, we discuss the use of nanoparticles, packed in a box-shaped or cuboid packed-bed device having 50 mm length, 5 mm width, and 3 mm bed-height for fast, high-resolution separation of proteins at low pressure. The low bed height allows the separation to be carried out at low pressure while the cuboid device reduces dispersion effects and thereby keeps the resolution high. Two different types of hydroxyapatite nanoparticles, i.e., needle-shaped (about 20 nm × 150 nm) and spherical (<200 nm) ones, were examined. The experimental results showed that while the needle-shaped nanoparticles were suitable for the separation of small proteins such as lysozyme, the spherical nanoparticles were better suited for separation of larger proteins such as bovine serum albumin and monoclonal antibody. The separation of the two proteins could be carried out in less than 2 min at a pressure lower than 0.8 MPa, using inexpensive chromatography devices a7nd systems, and without high pressure related problems such as frictional heating and on-column protein denaturation.

Effects of process parameters on the efficiency of chromatographic separations using a cuboid packed-bed device.

In recent papers we have proposed cuboid packed-bed devices as viable alternatives to preparative columns with low bed-height to diameter ratios that are typically used for biopharmaceutical purification processes. In this case study, we systematically examined operating parameters such as mobile phase flow rate, sample injection volume and feed protein concentration on the characteristics of flow-through and bound-and-eluted protein peaks. The current study was carried out using strong anion exchange media packed in a cuboid packed-bed device and its equivalent column, i.e. with identical volume and bed-height. Our experimental results showed that the cuboid packed-bed device outperformed its equivalent column at all conditions examined. However, the improvement in performance in flow-through experiments was more significant at lower flow rates, and when smaller sample volumes (i.e. less than 20% of bed volume) were injected. Also, the performance of the cuboid packed-bed was significantly better when a concentrated protein sample was injected using a small volume loop. In bind-and-elute experiments, the flow rate had a very significant impact on the performance of the cuboid packed-bed device, with better results being obtained at the lower flow rates examined. By choosing appropriate experimental conditions, significantly sharper peaks and thereby efficient separations could be achieved using the cuboid packed-bed device than with its equivalent column.

Feasibility study for high-resolution multi-component separation of protein mixture using a cation-exchange cuboid packed-bed device.

In recent papers we have discussed the optimization of design and operating conditions for cuboid packed-bed device for chromatographic separations. The efficiency metrics used in these studies included the number of theoretical plates per unit bed height as well as attributes of flow-through and eluted peaks. These studies were carried out using equivalent columns as benchmarks. The cuboid packed-bed devices consistently outperformed the columns in terms of the above metrics. The current study examines how well, or indeed if at all these superior efficiency metrics translate to superiority in multi-component protein separation. Cation exchange resin was examined in the current study using appropriate multi-component model protein system which was chosen with close isoelectric points to make the separation challenging. Effects of operating and experimental parameters such as flow rate, loop size and linear gradient length on separation performance were systematically investigated. Separation metrics examined included peak width, tailing factor, asymmetry factor and resolution of separated protein peaks. The results obtained showed that the cation exchange cuboid packed-bed device significantly outperformed its equivalent commercial column (e.g., the number of theoretical plates per unit bed height was 8636/m for the cuboid packed-bed device as opposed to 1480/m for the column at a flow rate of 0.5 mL/min). The difference in efficiency was particularly high at lower flow rate and when shorter gradients were employed. The results suggest that the cuboid packed-bed devices could potentially have promising application in preparative separations such as biopharmaceutical purifications.