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.

Enrichment and immobilization of macromolecular analytes on a porous membrane utilizing permeation drag.

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.

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.

Enrichment and immobilization of macromolecular analytes on a porous membrane utilizing permeation drag / 药物分析学报

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 macro-molecular 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 al-bumin (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 sophis-ticated detection methods.

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.

Ultrafast Separation and Analysis of Monoclonal Antibody Aggregates Using Membrane 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.

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.