Laterally-fed Membrane Chromatography Research Articles

Optimization of fluid flow in membrane chromatography devices using computational fluid dynamic simulations

Flow uniformity within the device is critically important in membrane chromatography. Recent studies have shown that the design of the device has a significant impact on flow uniformity, and thereby on separation efficiency. The main premise of this work is that computational fluid dynamics (CFD) could serve as a fast and inexpensive tool for preliminary optimization of the design of a membrane chromatography device. CFD also helps in identifying factors that affect flow uniformity. In this paper, CFD is used to compare the fluidic attributes of conventional membrane chromatography devices such as the stacked disc and radial flow devices with those of more recently developed ones such as the different versions of the laterally-fed membrane chromatography (LFMC) device. These are compared based on pulse tracer solute dispersion, which is a useful metric for measuring flow uniformity, and is thereby a good predictor of chromatographic separation performance. The poor separation performance typically observed with conventional membrane chromatography devices could be attributed to the high degree of solute dispersion within these devices. CFD is then used to analyze the impact of factors such as membrane aspect ratio, and channel dimensions on the performance of z2-laterally-fed membrane chromatography (z2LFMC) devices. The results discussed in the paper demonstrate that CFD could indeed serve as a powerful optimization and performance prediction tool for membrane chromatography.

A fast, efficient, and scalable method for purifying recombinant SARS-CoV-2 spike protein

Recombinant SARS-CoV-2 trimeric spike protein produced by mammalian cell culture is a potential candidate for a COVID-19 vaccine. However, this protein is much larger than most typical biopharmaceutical proteins and its large-scale manufacture is therefore challenging. Particularly, its purification using resin-based chromatography is difficult as the diffusive transport of this protein to and from its binding site within the pores of the stationary phase particles is slow. Therefore, very low flow rates need to be used during binding and elution, and this slows down the purification process. Also, due to its large size, the binding capacity of this protein on resin-based media is low. Membrane chromatography is an efficient and scalable technique for purifying biopharmaceuticals. The predominant mode of solute transport in a membrane is convective and hence it is considered better than resin-based chromatography for purifying large proteins. In this paper, we propose a membrane chromatography-based purification method for fast and scalable manufacture of recombinant SARS-CoV-2 trimeric spike protein. A combination of cation exchange z2 laterally-fed membrane chromatography and size exclusion chromatography was found to be suitable for obtaining a homogeneous spike protein sample from mammalian cell culture supernatant. The proposed method is both fast and scalable and could be explored as a method for manufacturing vaccine grade spike protein.

Fast and high-resolution fractionation of positional isomers of a PEGylated protein using membrane chromatography

The fractionation of positional isomers of a PEGylated protein is quite challenging as these have similar molecular weight, and only very slightly different surface charge. In this study, cation exchange z2 laterally-fed membrane chromatography (z2LFMC), which has been shown to be suitable for high-speed, high-resolution protein purification, was used to fractionate positional isomers of mono-PEGylated lysozyme. The performance of the z2LFMC device was compared with a commercial preparative cation exchange column having the same volume and ligand. PEGylated lysozyme purification experiments showed that while the positional isomers of mono-PEGylated lysozyme could not be satisfactorily resolved using the preparative commercial cation exchange column, almost baseline resolution of these could be achieved using the z2LFMC device. Moreover, the z2LFMC device-based process was faster by an order of magnitude. The results discussed in this paper demonstrate that z2LFMC is a superior alternative to column-based chromatography for challenging protein separations, such as the one discussed here, both in terms of speed and resolution.

Comparison of the performance of anion exchange membrane materials for adenovirus purification using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics used to treat a variety of diseases. Ion-exchange membrane chromatography is commonly used for virus purification, with the Mustang Q (Pall) and Sartobind Q (Sartorius) being two of the most widely used strong anion-exchange membranes. These membranes are made from different materials and have different pore sizes, and while previous studies have attempted to compare their performance in various virus purification applications, those studies have been hindered by the additional fact that these membranes are sold in different module formats. Thus, in order to establish a direct comparison of the capabilities and purification performance of anion exchange membrane materials, we first fabricated equivalent laterally-fed membrane chromatography (LFMC) devices containing either 1 mL of Mustang Q membrane or 1 mL of Sartobind Q membrane and then compared their performance based on residence times, the elution patterns of pre-purified biomolecules, and the recovery and purity of adenovirus from a cell lysate. Under the same linear gradient elution conditions, purified adenovirus eluted approximately one membrane volume earlier for the Sartobind Q membrane; a similar result was obtained during purification of adenovirus from infected suspension 293 cells. While the total recovery of adenovirus was the same for both membranes (~60%), the Sartobind Q membrane provided a much greater removal of DNA impurities in the virus containing elution fractions.

Purification of monoclonal antibody using cation exchange z2 laterally-fed membrane chromatography – A potential alternative to protein A affinity chromatography

Protein A affinity chromatography, which is the standard method for purifying monoclonal antibodies (mAbs) is expensive and involves elution at acidic pH, which could degrade the mAb. Moreover, it cannot be used to fractionate charge variants or to remove mAb aggregates. In this study, we examine the purification of Trastuzumab by cation-exchange z2 laterally-fed membrane chromatography (or z2LFMC). It has been shown that z2LFMC is suitable for carrying out high-speed, high-resolution protein purification. The results discussed in this paper demonstrate that the purity of Trastuzumab obtained by the z2LFMC process was comparable to that obtained by protein A chromatography (i.e., >95% with both methods), while the recovery was significantly greater with z2LFMC (90.4% as opposed to 81.7%). Also, the z2LFMC process was faster, did not require acidic pH conditions for elution, and the mAb was eluted in 2.24 bed volumes as opposed to 2.74 bed volumes with protein A chromatography. The mAb productivity obtained with the z2LFMC process was more than 3 times higher than that with the column-based purification process. The z2LFMC device is significantly cheaper than its equivalent protein A affinity column, and earlier studies have shown it to be suitable for separating mAb charge variants and aggregates. Therefore, the z2LFMC process could be evaluated as a cheaper and more efficient alternative to protein A affinity chromatography.

Donaldson Co Inc, USA

We examine the use of a z2 flow distribution and collection feature for enhancing the separation efficiency of a laterally-fed membrane chromatography (or LFMC) device. The new device is designated z2LFMC. Two devices were fabricated using two different ion-exchange membranes: strong anion exchange (Q) with 0.8-micron pore size, and strong cation exchange (S) with pore size in the 3–5 µm range. The number of theoretical plates per unit membrane bed height in these z2LFMC devices were higher than those reported for other membrane chromatography devices housing similar membranes, including the older versions of LFMC devices. The enhancement in separation efficiency is explained based on computational fluid dynamic (CFD) simulations. Model protein separation experiments showed that the z2LFMC device gave similar resolution as an equivalent resin-packed column, at 40-time greater flow rate than that used with the column. At a comparable flow rate, the resolution obtained with the z2LFMC device was significantly higher than that obtained with the equivalent resin-packed column. Therefore, the z2LFMC device combines high-speed with high-resolution. We demonstrate the suitability of the z2LFMC devices for carrying out fast and efficient biopharmaceutical separations through two case studies, i.e. the fractionation of monoclonal antibody charge variants, and monoclonal antibody aggregates separation.

Fast and high-resolution purification of a PEGylated protein using a z2 laterally-fed membrane chromatography device

PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In this study, we discuss the application of a newly developed z2 laterally-fed membrane chromatography (or z2LFMC) device for carrying out high-resolution purification of a PEGylated protein drug. The device used in the current study contained a stack of anion exchange (Q) membranes. The membrane bed-height of this z2LFMC device being small, it could be operated at very high flow rates, at relatively low back pressures. The primary goal was to speedily and efficiently separate a mono-PEGylated protein from impurities present in the PEGylation reaction mixture. A resin-based anion exchange column having the same ligand and bed-volume was used as the control device. The purification performance of the z2LFMC device and the control column were compared terms of resolution, recovery and purity. The z2LFMC device outperformed the control column in terms of every metric compared in this study. Higher purity (85.4% as opposed to 77.9%) and higher recovery (28% greater) of the target mono-PEGylated protein were obtained using the z2LFMC device at 20-time higher speed. These results clearly demonstrate that the z2LFMC device could be a faster and more efficient alternative to resin-based columns for purification of biopharmaceuticals.

A z2 laterally-fed membrane chromatography device for fast high-resolution purification of biopharmaceuticals.

We examine the use of a z2 flow distribution and collection feature for enhancing the separation efficiency of a laterally-fed membrane chromatography (or LFMC) device. The new device is designated z2LFMC. Two devices were fabricated using two different ion-exchange membranes: strong anion exchange (Q) with 0.8-micron pore size, and strong cation exchange (S) with pore size in the 3-5 µm range. The number of theoretical plates per unit membrane bed height in these z2LFMC devices were higher than those reported for other membrane chromatography devices housing similar membranes, including the older versions of LFMC devices. The enhancement in separation efficiency is explained based on computational fluid dynamic (CFD) simulations. Model protein separation experiments showed that the z2LFMC device gave similar resolution as an equivalent resin-packed column, at 40-time greater flow rate than that used with the column. At a comparable flow rate, the resolution obtained with the z2LFMC device was significantly higher than that obtained with the equivalent resin-packed column. Therefore, the z2LFMC device combines high-speed with high-resolution. We demonstrate the suitability of the z2LFMC devices for carrying out fast and efficient biopharmaceutical separations through two case studies, i.e. the fractionation of monoclonal antibody charge variants, and monoclonal antibody aggregates separation.

Integrated development of enzymatic DNA digestion and membrane chromatography processes for the purification of therapeutic adenoviruses

High titer and purity levels are key requirements for virus-based cancer and gene therapy biopharmaceuticals. However, the task of removing enough host-cell DNA from therapeutic viruses in order to comply with the FDA’s 10 ng/dose limit is particularly challenging. In a previous study, we demonstrated the advantages of using laterally-fed membrane chromatography (LFMC) for adenovirus purification. Although this approach achieved >90% DNA removal, significant amounts of DNA remained in the product due to the poor performance of a pre-LFMC DNA digestion step. In the present study, we attempt to improve upon this outcome by employing an integrated approach to process development that examines the interactions between different downstream steps (i.e., clarification, enzymatic DNA digestion, and membrane chromatography (MC)). First, we identified the most efficient process sequence involving a clarification step followed by a DNA digestion step, as well as three endonucleases (Benzonase®, Denarase®, and Turbonuclease™) that perform similarly with respect to DNA digestion. Next, a factorial design of experiments (DOE) study was used to evaluate how enzyme (Benzonase and Denarase) concentration and incubation time impact the resulting DNA and virus concentrations. Since Denarase showed slightly better efficiency, it was used along with a subset of the DOE conditions to evaluate the removal of DNA via MC with Sartobind Q 96-well filter plates. The lowest amount of DNA per dose was achieved using MC in conjunction with feed that had been digested with 10 U/mL of Denarase for 4 h; as such, this approach was used to prepare lysates, which were then purified using an LFMC device containing 1 mL of Sartobind Q membrane. This process enabled a virus-recovery rate of 73% and residual DNA levels of 77 ng/dose. Ultimately, the proposed integrated process development approach resulted in an approximately 80-fold improvement in DNA removal.

Performance Comparison of a Laterally-Fed Membrane Chromatography (LFMC) Device with a Commercial Resin Packed Column.

The use of conventional membrane adsorbers such as radial flow devices is largely restricted to flow-through applications, such as virus and endotoxin removal, as they fail to give acceptable resolution in bind-and-elute separations. Laterally-fed membrane chromatography or LFMC devices have been specifically developed to combine high-speed with high-resolution. In this study, an LFMC device containing a stack of strong cation exchange membranes was compared with an equivalent resin packed column. Preliminary characterization experiments showed that the LFMC device had a significantly greater number of theoretical plates per metre than the column. These devices were used to separate a ternary model protein mixture consisting of ovalbumin, conalbumin and lysozyme. The resolution obtained with the LFMC device was better than that obtained with the column. For instance, the LFMC device could resolve lysozyme dimer from lysozyme monomer, which was not possible using the column. In addition, the LFMC device could be operated at lower pressure and at significantly higher flow rates. The devices were then compared based on an application case study, i.e., preparative separation of monoclonal antibody charge variants. The LFMC device gave significantly better separation of these variants than the column.

Purification of therapeutic adenoviruses using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics with the potential to treat cancer, genetic, cardiovascular and other diseases. Their commercial success depends on cost-effective purification processes. Membrane chromatography processes offer great potential for virus purification, however conventional devices are known to suffer from issues related to resolution and sample dilution. Laterally-fed membrane chromatography (LFMC) overcomes these issues by minimizing the dead-volume and establishing a uniform flow distribution pattern within the device. While the efficiency of LFMC has been previously demonstrated for protein purification studies, here we present a direct comparison of the performance of a LFMC device and a conventional radial flow device (both containing 1 mL of the same strong anion-exchange membrane) for the purification of adenoviruses. Using a gradient elution strategy, a 74% virus recovery with 4% of residual amount of total protein and 1% residual amount of total DNA was obtained using the LFMC device; for the radial flow device, only 20% virus recovery was achieved for a similar level of purity. Using a stepwise elution strategy, close to 100% virus recovery with 10% residual protein and DNA was achieved with both devices. However, the product was at least 50% more dilute with the radial flow device. Overall, this study demonstrates the considerable benefits of using LFMC over conventional membrane chromatography devices for purifying virus-based biotherapeutics.

Computational fluid dynamic (CFD) simulation of laterally-fed membrane chromatography

Currently, most bioseparation processes used for purification of bio-pharmaceutical products such as vaccines, monoclonal antibodies, and enzymes use resin-based column chromatography. Resolution and productivity are two of the major factors that should be optimized in any purification process. While column chromatography techniques give high-resolution and consequently very high product quality, they are slow, leading to low-productivity and high production cost. One of the alternatives to resin-based columns is membrane chromatography, where a stack of membrane sheets is used as chromatographic media. Membrane chromatography is a fast and scalable technique; however, it is not commonly used for some of the critical purification steps due to its low-resolution capability. A technique newly developed in our group called laterally-fed membrane chromatography (or LFMC) combines high-resolution separation with high-productivity. It is, therefore, suitable for rapid, multicomponent protein purification. LFMC not only gives higher resolution than other membrane chromatography devices, it gives comparable resolution to that obtained with equivalent resin-based packed bed columns, even at significantly higher flow rates, as demonstrated in our previous work. This paper examines the system fluid dynamics in an LFMC device using computational fluid dynamics (CFD) simulations and explains the reasons behind its superior separation attributes.

Comparison of membrane chromatography devices in laboratory-scale preparative flow-through separation of a recombinant protein

ABSTRACTRadial-flow membrane chromatography is widely used for biopharmaceutical purification, particularly for separations carried out in the flow-through mode. In this article, we propose laterally fed membrane chromatography (LFMC) as an alternative to radial-flow membrane chromatography. LFMC has already been shown to be suitable for carrying out fast, high-resolution separations. In this study, we compare the performance of a radial-flow cation exchange membrane device with its equivalent LFMC device for flow-through separation of SP1, a recombinant protein that is currently under development by Sanofi Pasteur as a vaccine candidate. The flow-through of SP1 was monitored by UV absorbance while the breakthrough of the impurities such as host cell proteins was measured by analysing collected samples by Western blotting followed by densitometric scanning. The results obtained clearly demonstrated multiple-attribute superiority of the LFMC device in the above separation.

Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster.

Preparative separation of monoclonal antibody aggregates by cation-exchange laterally-fed membrane chromatography.

Cation exchange (CEX) chromatography is widely used for large-scale separation of monoclonal antibody (mAb) aggregates. The aggregates bind more strongly to CEX media and hence elute after the monomeric mAb in a salt gradient. However, monomer-aggregate resolution that is typically obtained is poor, which results in low product recovery. In the current study we address this challenge through the use of cation-exchange laterally-fed membrane chromatography (LFMC). Three different LFMC devices, each containing a bed of strong cation-exchange (S) membranes were used for preparative-scale removal of mAb aggregates. Trastuzumab (IgG1) biosimilar derived from human embryonic kidney 293 (293) cells was used as the primary model mAb in our study. The other mAbs investigated were Chinese hamster ovary (CHO) cell line derived Alemtuzumab (Campath-1H) and a heavy chain chimeric mAb EG2-hFc. In each of these case-studies, aggregates were well-resolved from the respective monomer. The separated and collected monomer and aggregate fractions were analyzed using techniques such as hydrophobic interaction membrane chromatography (HIMC), native polyacrylamide gel electrophoresis (or PAGE), and size-exclusion high-performance liquid chromatography (SE-HPLC). The high efficiency of separation obtained in each case was due to a combination of the small membrane pore size (3-5μm), and the use of LFMC technology, which has been shown to be suitable for high-resolution, multi-component protein separations. Also, the LFMC based separation processes reported in this study were more than an order of magnitude faster than equivalent resin-based, cation exchange chromatography.

Using a box instead of a column for process chromatography

Columns with relatively short bed-height to diameter ratios are frequently used for process-scale chromatography applications such as biopharmaceutical purification. Non-uniform flow distribution within such columns could result in broad and poorly resolved eluted peaks, which could in turn affect purity, recovery and productivity of the process. Different strategies centered on improved column header design have been proposed for addressing this problem. This paper describes a radically different approach, i.e. the use of a chromatography box (or chromato-box) instead of a column, for addressing the challenges posed by flow mal-distribution in process-scale, packed-bed chromatography devices. The design of the chromatography box devices used in this study is based on a laterally-fed membrane chromatography (or LFMC) device, that has been described and discussed in several recent papers. The performances of two chromatography box devices were compared with their equivalent columns in terms of sharpness and asymmetry of flow-through and eluted peaks, number of theoretical plates per metre, and peak resolution in binary and ternary protein separations. In each of the above comparisons, the chromatography box devices performed better than their equivalent columns, clearly indicating their potential as an alternative in process-scale chromatography applications.

High-resolution, preparative purification of PEGylated protein using a laterally-fed membrane chromatography device.

We discuss the use of a laterally-fed membrane chromatography (or LFMC) device for single-step purification of mono-PEGylated lysozyme. Recent studies have shown such LFMC devices to be suitable for high-resolution, multi-component separation of proteins in the bind-and-elute mode. The device used in this study contained a stack of rectangular cation-exchange membranes having 9.25mL bed volume. PEGylation of lysozyme was carried out in batch mode using 5kDa methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) in the presence of sodium cyanoborohydride as reducing agent. Membrane chromatographic separation was carried out at 1.62 membrane bed volumes per minute flow rate, in the bind-and-elute mode. When a salt gradient was applied, the higher PEGylated forms of lysozyme (i.e. the byproducts) eluted earlier than mono-PEGylated lysozyme (the target product), while lysozyme eluted last. Under elution conditions optimized for resolution and speed, the separation could be carried out in less than 15 membrane bed volumes. High purity and recovery of mono-PEGylated lysozyme was obtained. The resolution of separation of mono-PEGylated lysozyme obtained under the above condition was comparable to that reported in the literature for equivalent cation-exchange resin columns while the flow rate expressed in bed volumes/min was 21.7 times higher. Also, the number of theoretical plates per meter was significantly higher with the LFMC device. Therefore the LFMC based purification process discussed in this paper combined high-productivity with high-resolution.

On the workings of laterally-fed membrane chromatography

Membrane chromatography has significant potential for use in biopharmaceutical purification processes. However, currently available membrane chromatography devices give poor resolution and therefore their use is restricted to polishing applications, typically carried out in the flow-through mode. Laterally-fed membrane chromatography (or LFMC) was developed specifically for carrying high-resolution multi-component protein separations. Recent studies have shown that resolution obtained with LFMC is comparable to that obtained with equivalent resin-based packed bed chromatography. Therefore, LFMC combines high-resolution with high-productivity, a highly desirable attribute in any purification technique. In this paper we explain the workings of LFMC based on theoretical and experimental analysis of hydraulic flow-path and residence time distribution. Factors likely to affect efficiency of LFMC are discussed using a simple electrical circuit analogy.

High-resolution protein separation using a laterally-fed membrane chromatography device

Radial-flow membrane chromatography devices which are used for flow-through separation are generally unsuitable for bind-and-elute chromatography, particularly where multiple components need to be separated. We discuss a laterally-fed membrane chromatography device, suitable for high-resolution, multi-component protein separation in the bind-and-elute mode. In the current study, a stack of cation exchange membranes was housed within the membrane device and its performance was compared with an equivalent commercial radial-flow device having the same membrane bed volume and bed height. Tracer experiments were carried out using sodium chloride solution to compare their residence time distributions. The laterally-fed device showed superior flow distribution characteristics, which could be attributed to a lower variability in solute-flow path-lengths, and a smaller dead volume. Single protein bind-and-elute experiments carried out using lysozyme showed that the peaks obtained with the laterally-fed device were significantly sharper and more symmetrical. Excellent separation of three model proteins ovalbumin, conalbumin and lysozyme demonstrated that the laterally-fed membrane chromatography device was indeed suitable for carrying out high-resolution multi-component protein purification. These proteins could be fractionated in about 10 membrane bed volumes using the laterally-fed device as opposed to 25 bed volumes with the radial-flow device. The design of the laterally-fed device is simple and its flat shape gives it significantly lower footprint and offers additional advantages such as stackability and ease of multiplexing.