Purification of rabbit polyclonal immunoglobulin G using anion exchangers (original) (raw)
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Journal of the American Chemical Society, 2009
This paper describes a method for the purification of monoclonal antibodies (rat anti-2,4dinitrophenyl IgG: IgG DNP ; and mouse anti-digoxin IgG: IgG Dgn) from ascites fluid. This procedure (for IgG DNP) has three steps: i) precipitation of proteins heavier than immunoglobulins with ammonium sulfate; ii) formation of cyclic complexes of IgG DNP by causing it to bind to synthetic multivalent haptens containing multiple DNP groups; iii) selective precipitation of these dimers, trimers, and higher oligomers of the target antibody, followed by regeneration of the free antibody. This procedure separates the targeted antibody from a mixture of antibodies, as well as from other proteins and globulins in a biological fluid. This method is applicable to antibodies with a wide range of monovalent binding constants (0.1 μM to 0.1 nM). The multivalent ligands we used (derivatives of DNP and digoxin) isolated IgG DNP and IgG Dgn from ascites fluid in yields of > 80% and with > 95% purity. This technique has two advantages over conventional chromatographic methods for purifying antibodies: i) it is selective for antibodies with two active Fab binding sites (both sites are required to form the cyclic complexes) over antibodies with one or zero active Fab binding sites; ii) it does not require chromatographic separation. It has the disadvantage that the structure of the hapten must be compatible with the synthesis of bi and/or trivalent analogs.
Journal of microbiology, biotechnology and food sciences
Monoclonal antibodies (mAbs) have become essential analytical tools for biomedical and veterinary diagnosis and the analytical release of other biomolecules due to their high specificity and reproducibility. The mAbs generated in mice contain murine serum albumin (MSA) as the main potential contaminant; this molecule interferes in a large number of tests in which these mAbs are required. For this reason, the development of analytical methods to identify and quantify MSA traces plays an important role in the quality of purified mAb. In this study, an anti-MSA polyclonal antibody (pAb) was generated in rabbit with titers of 1:2 700 000 and purified by nProtein A affinity chromatography with purity greater than 90%. The purified antibody was conjugated to horseradish peroxidase (HRP) by the m-periodate method with an optimal working dilution of 1:64 000 in direct ELISA. Two applications were evaluated for the analytical release of mAbs: identity test by Western Blot and direct ELISA, a...
Journal of Immunological Methods, 2002
Transferrin and albumin are often present in immunoglobulin G (IgG) concentrates and are considered as impurities. Therefore, it is important to determine their concentration in order to obtain a well-characterized biological product. Here, we describe their determination based on conjoint liquid chromatography (CLC). The established method combines two different chromatographic modes in one step: affinity and ion-exchange chromatography (IEC) combined in one column. Therefore, two CIM Protein G and one CIM quaternary amine (QA) monolithic disks were placed in series in one housing forming a CLC monolithic column. Binding conditions were optimized in a way that immunoglobulins were captured on the CIM Protein G disks, while transferrin and albumin were bound on the CIM QA disks. Subsequently, transferrin and albumin were eluted separately by a stepwise gradient with sodium chloride, whereas immunoglobulins were released from the Protein G ligands by applying low pH. A complete separation of all three proteins was achieved in less than 5 min. The method permits the quantification of albumin and transferrin in IgG concentrates and has been successfully validated. D
Journal of Chromatography B, 2013
The purification of antibodies by precipitating impurities using Polyethylene Glycol (PEG) was assessed with the objective of developing a two chromatography column purification process. A PEG precipitation method was evaluated for use in the industrial purification of recombinant monoclonal antibodies (MAbs). Effective and robust precipitation conditions including PEG concentration, pH, temperature, time, and protein concentration were identified for several different MAbs. A recovery process using two chromatography steps in combination with PEG precipitation gave acceptable yield and purity levels for IgG1 and IgG4 antibodies with a broad range of isoelectric points (pI). PEG precipitation removed host cell proteins (HCPs), high molecular weight species (HMWS), leached Protein A ligand, and host cell DNA to acceptable levels when run under appropriate conditions, and some endogenous virus removal was achieved.
To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100ppm and <10ppb respectively relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation a) Harvest treatment b) Protein-A Chromatography and c) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification.
Purification of Antibodies: Diethylaminoethyl (DEAE) Chromatography
Cold Spring Harbor Protocols, 2019
Because IgG from most species (other than rodents) tends to have an isoelectric point around neutral, two approaches can be used when separating IgG using diethylaminoethyl (DEAE) resins. When serum containing antibodies is applied to DEAE at a slightly acidic pH, the IgG flows through the column while most other serum proteins bind to the DEAE. This method is best performed using a batch method. The DEAE beads can be kept in a disposable syringe containing a polypropylene frit, a glass reactor containing a coarse-sintered glass frit, or other suitable vessel. If the antibody solution is adjusted to a basic pH of 8–8.5, then IgG binds to the DEAE resin. After washing the column, the antibody is eluted by adding a buffer of increasing ionic strength to the column. Prepacked columns of many sizes are available for the isolation of antibodies by DEAE chromatography. Alternatively, DEAE medium can be swelled or prepped according to the manufacturer's instructions and a column can be...
OALib, 2014
The purified Immunoglobulin G (IgG) is effectively used in passive immunization. There are various methods to isolate the IgG from serum, with its own advantages and disadvantages. In the current study, a comparative efficacy of the affinity chromatography using Protein A-Sepharose and ion exchange chromatography using DEAE Sephadex A50 was done with regard to yield and purity of IgG. Both methods were found equally effective in isolation of pure IgG with similar recovery, indicating that researcher can use either method to purify IgG depending on available resources.