Pedram Madadkar

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.

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.

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.

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

ABSTRACT Radial-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.

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.