Use Of Membrane Chromatography Research Articles

Ultrahigh-speed, ultrahigh-resolution preparative separation of protein biopharmaceuticals using membrane chromatography.

This paper discusses ultrahigh-speed, ultrahigh-resolution preparative protein separation using an in-house designed membrane chromatography device. The performance of the membrane chromatography device was systematically compared with an equivalent resin-packed preparative column. Experiments carried out using model proteins showed that membrane chromatography gave more than four times greater resolution than the preparative column, while at the same time being more than 19 times faster. Membrane chromatography was therefore a better option, not only in terms of higher productivity but also in terms of higher product purity. Membrane chromatography was also superior in terms of resolving and presenting tracer impurity peaks in the chromatogram. Experiments carried out using monoclonal antibody samples showed that membrane chromatography was suitable for ultrahigh speed, and ultrahigh resolution fractionation of charge variants. This paper highlights and explains the need for proper device design for enabling the use of membrane chromatography for the efficient purification of protein biopharmaceuticals.

Comparison of Cutinase Separation in Different Chromatographic Media

Cutinase is a hydrolytic enzyme that has both properties of lipase and esterase, thus finding its use in many areas. Previous studies have investigated both upstream and downstream processes for cutinase production from microbial source. However, no study has yet to address the use of membrane chromatography for cutinase purification, which is more favourable in terms of process resolution and product throughput as compared to the conventional gel chromatography. Hydrophobic interaction was chosen as the separation mechanism for cutinase purification in this study. The optimisation of cutinase purification in two different types of chromatographic media; conventional packed-gel and membrane matrix, were pre-determined by the best compromise between the recovery and purity of the purified cutinase. It was found that the optimised condition were of pH 4.0 and 1.0 M ammonium sulfate for the conventional column (50% recovery, 4.8-fold purity) and pH 6.0 with 1.5 M ammonium sulfate for the membrane–matrix column(87% recovery, 30-fold purity). Preferential interaction analysis was used to describe the protein chromatographic behaviour in each chromatographic media. Graph of natural algorithm of protein retention data to the function of salt concentration at pH 4.0 and 6.0 for each column were plotted. It was found that at the optimum pH condition for gel-packed column, a small amount of ammonium sulfate was sufficient to achieve maximum cutinase recovery and purity since the effect of salt at that particular pH were less significant. Consequently, the number of released water molecules were calculated and it was observed that for membrane column, larger number of water was released at pH 6.0 illustrating more protein were bounded to the stationary phase, thus explaining the optimum pH condition of the particular column.

Fractionation of different PEGylated forms of a protein by chromatography using environment-responsive membranes

PEGylation of therapeutic proteins can enhance their efficacy as biopharmaceuticals through increased stability and hydrophilicity, and decreased immunogenicity. A site-specific PEGylated protein (e.g. mono-PEGylated at N-terminus) is frequently desirable as a product. However, multiple-PEGylated forms are frequently produced as byproducts. In this paper we discuss the fractionation of the different PEGylated forms of a protein by hydrophobic interaction chromatography using a stack of hydrophilized PVDF membrane, which has been shown to be environment responsive, as stationary phase. With the model protein examined in this study (i.e. lysozyme), the apparent hydrophobicity in the presence of a lyotropic salt increased with the degree of PEGylation. Based on this, unmodified lysozyme and its mono-, di- and tri-PEGylated forms could each be resolved into separate chromatographic peaks. Such fractionation was not feasible using conventional hydrophobic interaction chromatography using a butyl column. The use of membrane chromatography also ensured that the fractionation was fast and hence suitable for analytical applications such as product purity determination and monitoring of the extent of PEGylation reactions.

Anion-exchange membrane chromatography for purification of rotavirus-like particles

Anion-exchange chromatography is one of the separation processes of choice for the recovery/purification of proteins and complex bio-structures as viruses or virus-like particles (VLPs), e.g., rotavirus-like particles (RLPs) representing a potential biopharmaceutical, where a safer vaccine technology is endeavored. Purification strategies of complex bioparticles are as yet rather empirical. The breakthrough pursued hereby was to gain theoretical and predictive knowledge on the anion-exchange membrane chromatography processing of this type of products. Currently, the use of membrane chromatography is expanding due to its scalability, robustness and disposable nature, allowing high fluxes, rapid processing, little buffer consumption and a validation-free environment. A novel matrix, Sartobind™ D membrane adsorber from Sartorius, was used and thoroughly studied for the adsorption of VLPs. Screening studies showed the sensitive effect of the ionic strength and the pH of the equilibration buffer on product final recovery. Steric mass action (SMA) model formulation was implemented for the prediction of the elution profile of purified RLP pulses. Experimental data fitting gave good model validation therefore yielding insights on the fundamental understanding of the ion-exchange separation mechanism of virus-like particles and, potentially, for other similar structures. Anion-exchange membrane chromatography was finally used in a larger scale downstream process, confirming that rotavirus VLPs can be reproductively purified to clinical grade at 46% global recovery yield, concomitant with nearly 100% removal of host bulk DNA and approximately 98% of host cell proteins.

182. An Effective and Valuable Process Using Disposable Membrane-Based Technologies for Pure Plasmid DNA Isolation and Purification: Throughput, Quality, and Cleaning Validation Advantages

There is high demand for high purity plasmid DNA for use in the fields of gene therapy and DNA vaccination. A need also exists for a cost-effective process that significantly reduces the time and expense of current plasmid production technologies. Our method involves purifying plasmid DNA from lysed bacterial cells using only disposable membrane technologies. We have previously demonstrated the use of an automated lysis machine, consisting of a high-shear mixing device and an air column. This method is highly cost-effective for production runs of commercial-scale plasmid. We present here an overview of the downstream purification parameters, which also supports cost-effective and efficient processing. Additionally, we have demonstrated that the use of membrane chromatography simplifies compliance with ever-evolving regulatory standards in areas such as cleaning validation and process control.