Deqiang Yu

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes.

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

Purification of PEGylated Protein Using Membrane Chromatography

N-terminus-specific PEGylation was used to produce mono-PEGylated lysozyme. However, some di- and tri-PEGylated proteins were also produced due to side chain reaction. The reaction products were characterized by chromatographic and electrophoretic methods. Commercial cation exchange membrane Sartobind S was used for chromatographic purification of PEGylated lysozyme, the basis of separation being the shielding of protein charge by PEG. Using the membrane chromatographic method, lysozyme and mono-, di-, and tri-PEGylated lysozyme could be resolved into separate peaks. Increasing the superficial velocity during chromatographic separation from 24 cm/h to 240 cm/h increased both protein binding capacity and resolution due to enhancement of protein mass transfer coefficient.

Integrated solid-phase synthesis and purification of PEGylated protein.

We describe an integrated method for solid-phase protein PEGylation and the purification of mono-PEGylated protein thus synthesized. Lysozyme was used as model protein in this study. Methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) was first immobilized on a stack of microporous hydrophobic interaction membranes housed in a module. The membrane-bound m-PEG propionaldehyde was then contacted with lysozyme solution, which also contained sodium cyanoborohydride as a reducing agent. The PEGylated lysozyme thus synthesized remained attached to the membrane, whereas unreacted protein could easily be removed from the module. PEGylated protein was then eluted from the membrane in a partially purified form using salt-free buffer. Two separate steps were thus integrated into a single process: protein PEGylation, followed by purification of mono-PEGylated protein. This solid-phase method is likely to be suitable for PEGylating any protein because it is based on the immobilization of the activated PEG and not the protein being PEGylated.

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.

Integrated fragmentation of human IgG and purification of Fab using a reactant adsorptive membrane bioreactor separator system

This article discusses an integrated separation–reaction–separation scheme for producing Fab fragment directly from human immunoglobulin G (hIgG) present in serum feed. The novel reactant adsorptive membrane bioreactor separator (or RAMBS) system used in the current study consisted of a stack of microporous adsorptive membranes held within a temperature controlled module. The membrane stack, in the presence of salt, selectively and reversibly adsorbed hIgG by hydrophobic interaction while allowing most other serum proteins to flow through. The bound hIgG was then fragmented by pumping a solution of papain through the reactor at controlled temperature and flow rate. The salt concentration and pH for reaction and separation were systematically optimized using pure hIgG as reactant. The Fab fragment was separated from undigested hIgG and other byproducts such as Fc fragment based on their differences in hydrophobicity. Under optimal conditions, Fab was obtained in the reaction flow through while the other proteins remained bound to the membrane, these being subsequently eluted by lowering the salt concentration. The RAMBS system in addition to being convenient from process integration and intensification points of view also showed higher catalytic efficiency of papain in comparison to that in liquid phase reactions. Biotechnol. Bioeng. 2009; 104: 152–161

Membrane bioreactor separator system for integrated IgG fragmentation and Fab purification

This paper discusses a membrane based bioreactor system for producing pure Fab from human IgG. The bioreactor consisted of a stack of microporous anion-exchange membrane discs housed in a temperature controlled module. IgG was adsorbed on the membrane followed by its fragmentation with papain under optimized conditions. Fab was recovered in the reaction flow through while other fragments remained membrane bound and were subsequently eluted using high salt concentration buffer. By using the membrane bioreactor-separator system the overall process for producing Fab was simplified and high product purity and recovery were achieved. The pH of the feed solution had a significant effect on Fab recovery. The rate of IgG fragmentation by papain observed with the membrane bioreactor was about three times higher than that in an equivalent liquid phase reaction.

Enzymatic fragmentation of cation exchange membrane bound immunoglobulin G

Immunoglobulin G (IgG) was immobilized on a stack of microporous cation‐exchange membranes and pulsed with pepsin solution. Fc fragment and its sub‐fragments thus produced were removed along with the reaction flow‐through, whereas F(ab′)2 which remained membrane bound could subsequently be eluted in a pure form using salt. The extent of IgG fragmentation and the apparent reaction rate constant were both significantly higher than in equivalent liquid phase reaction, presumably due to a combination of mass transport, steric, and substrate concentration effects. This approach of using a membrane surface as molecule cutting board could be attractive in niche applications such as integrated enzymatic reaction and purification processes involving macromolecular substrates.