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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.

Disc membrane chromatography modules are widely used for laboratory-scale protein separations. These have high radial to axial aspect ratios, with the inlet and the outlet being located at the centre. This results in a huge variability in flow path lengths. Also, the radial velocity in the feed side header decreases very significantly in an outward direction, while that in the permeate side header increases in an inward direction. Therefore, the flow paths closer to the axis of the membrane disc have higher average velocity compared to those closer to the periphery. Consequently, the solute residence time distribution within the disc membrane chromatography module is very large, which results in broad flow-through and eluted peaks and early breakthrough of bound solutes. In this paper, we discuss a simple but effective solution to this problem which involves the incorporation of a flow directing layer (FDL) within the membrane module. The FDL could be placed either in the front or at the rear of the membrane disc. The presence of FDL results in equalization of path lengths within the module, leading to narrowing of residence time distribution, and thereby improvement in separation efficiency. Modules housing strong cation exchange membrane discs having two different bed volumes of 0.4 mL (40 mm diameter) and 4.7 mL (60 mm diameter) were designed and fabricated in-house. The impact of using FDL on both the front side (F-FDL) and the rear side (R-FDL) was investigated. The separation efficiency of the membrane modules was assessed using salt tracer experiments, breakthrough protein binding, and ternary protein separation carried out in the bind-and-elute mode. The results confirmed that the use of the FDL significantly enhanced the separation efficiency. In addition to membrane chromatography, this approach of using an FDL could be extended to other membrane disc based modules such as in-line and syringe filters and membrane reactors.

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.

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.

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.

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.

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.

PEGylation is increasingly being utilized to enhance the therapeutic efficacy of biopharmaceuticals. Various chemistries and reaction conditions have been established to synthesize PEGylated proteins and more are being developed. Both the extent of conversion and selectivity of protein PEGylation are highly sensitive to process variables and parameters. Therefore, microfluidic-based high-throughput screening platforms would be highly suitable for optimization of protein PEGylation. As part of this study, a poly-dimethylsiloxane-based continuous flow microreactor system was designed and its performance was compared head-to-head with a batch reactor. The reactants within the microreactor were contacted by passive micromixing based on chaotic advection generated by staggered herringbone grooves embedded in serpentine microchannels. The microreactor system was provided with means for on-chip reaction quenching. Lysozyme was used as the model protein while methoxy-polyethylene glycol-(CH(2))(5)COO-NHS was used as the PEGylation reagent. Full mixing was achieved close to the microreactor inlet, making the device suitable for protein PEGylation. The effect of mixing type, i.e., simple stirring versus chaotic laminar mixing on PEGylation, was investigated. Higher selectivity (as high as 100% selectivity) was obtained with the microreactor while the conversion was marginally lower.

Enrichment and immobilization of analytes by chemical bonding or physical adsorption is typically the first step in many commonly used analytical techniques. In this paper, we discuss a permeation drag based technique as an alternative approach for carrying out location-specific immobilization of macromolecular analytes. Fluorescein isothiocyanate (FITC) labeled macromolecules and their complexes were enriched near the surface of ultrafiltration membranes and detected by direct visual observation and fluorescence imaging. The level of macromolecule enrichment at the immobilization sites could be controlled by manipulating the filtration rate and thereby the magnitude of permeation drag. Higher enrichment as indicated by higher fluorescence intensity was observed at higher filtration rates. Also, larger macromolecules were more easily enriched. The feasibility of using this technique for detecting immunocomplexes was demonstrated by carrying out experiments with FITC labeled bovine serum albumin (FITC-BSA) and its corresponding antibody. This permeation drag based enrichment technique could potentially be developed further to suit a range of analytical applications involving more sophisticated detection methods. Keywords: Macromolecule, Enrichment, Immobilization, Concentration polarization, Permeation drag, Immunoassay, Membrane, Ultrafiltration

Module design is of critical importance in membrane chromatography as the efficiency of separation is highly dependent on fluid flow distribution and collection within the membrane device. We discuss a novel, laterally-fed module, designed specifically for flat-sheet membrane chromatography. The performance of the novel module was compared with that of a conventional, centrally-fed, circular membrane module. Experiments were carried out with both devices using anion-exchange membrane sheets having the same surface area and thickness, and thereby the same bed volume. Tracer experiments using either a dye or a protein (lysozyme) under non-binding condition clearly indicated superior flow distribution and collection within the novel module. This could be attributed to greater uniformity in solute flow path length. The protein binding capacities of membrane sheets of identical surface area and bed volume housed in the novel and conventional modules were compared in the breakthrough and pulse modes, using bovine serum albumin (or BSA) as the model adsorbed protein. The breakthrough experiments showed that at the same experimental conditions, the 1% breakthrough binding capacity of the membrane housed in the novel module was 5.12 times higher than that housed in the conventional module. Moreover, flow-through and elution peaks obtained with the novel membrane module were significantly sharper and more symmetrical, with lower peak width.

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 (3–5 μm), 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.

We discuss a method for rapid and cost-effective analysis of monoclonal antibody (mAb) aggregates. Hydrophobic interaction membrane chromatography, which was previously shown to be highly suitable for such separation and analysis, was used in a recently developed format referred to as laterally fed membrane chromatography (or LFMC). A stack of rectangular polyvinylidene fluoride (or PVDF) membranes having 0.22 μm pores housed within a modified analytical-scale LFMC device was used for analyzing aggregate types and content in different monoclonal antibody samples. High-resolution separations could be achieved in less than 1.5 min, this being faster than other currently available techniques such as size exclusion ultraperformance liquid chromatography (SE-UPLC). Moreover, the operating pressure was less than 200 kPa, which eliminated the need for an expensive high-pressure pump and chromatography system. The resolution obtained using the LFMC was comparable to that obtained using SE-UPLC. The effect of design variations such as change in dead volume and pillar size within the lateral channels within the LFMC device was also examined.

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.