Feasibility Study for the Fractionation of the Major Human Immunoglobulin G Subclasses Using Hydrophobic Interaction Membrane Chromatography

Abstract

Human immunoglobulin G (IgG) consists of four subclasses, each having specific biological functions and physical properties. Fractionation of these subclasses is challenging, both at analytical and preparative scales. This paper examines the feasibility of separating the major IgG subclasses, i.e., IgG1 and IgG2, by hydrophobic interaction membrane chromatography using environment-responsive membranes. These were resolvable as separate peaks at appropriate eluting conditions. This method could potentially be developed further into a rapid and robust IgG subclass profiling technique, suitable for diagnostic applications. This method could also be used to produce specific subclass enriched IgG.

Cellulose Nanofibrous Membranes Modified with Phenyl Glycidyl Ether for Efficient Adsorption of Bovine Serum Albumin

“…These advantages make membrane chromatography much more attractive these years. Polyvinylidene fluoride microporous membranes applied for purification of monoclonal antibody, chimeric heavy chain monoclonal antibody, human plasma proteins, mono-PEGylated lysozyme, immunoglobulin G and its subclasses have been reported by Ghosh and coworkers, implying that membranes could be a kind of cost-effective media for protein HIC with high efficiency [7][8][9][10][11][12][13][14][15]. Moreover, filter papers modified with polyethylene glycol, poly-(N-isopropylacrylamide)butyl-acrylate or poly (N-vinyl caprolactam) were also developed as HIC media [6,16,17].…”

Poly(ethylene glycol)‐based monolithic capillary columns for hydrophobic interaction chromatography of immunoglobulin G subclasses and variants

“…The separation and quantification of these subclasses is therefore extremely important, especially in cases where there exists a subclass deficiency but normal amounts of total IgG . Further complications arise as each of the subclasses has additional variants including both a κ and a λ variant, which signifies a difference in the light chain , in contrast to the subclasses themselves, which have the greatest structural difference in what is known as the hinge region . These variants also have clinical importance, for example the κ to λ light chain ratio has previously been used as a clinical criterion for non‐Hodgkin B‐cell lymphomas, where this ratio differs from normal .…”

“…The separation and ultimate purification of IgG subclasses often involves the use of protein A based affinity chromatography. For example, this technique has been found suitable for the separation of IgG3 from total IgG, due to the inability of IgG3 to form a complex with protein A . However, this technique is expensive, prone to ligand leakage from the sorbent, and under the elution conditions, the formation of aggregates is sometimes observed.…”

“…However, this technique is expensive, prone to ligand leakage from the sorbent, and under the elution conditions, the formation of aggregates is sometimes observed. In addition, complete separation of these subclasses has yet to be demonstrated solely by using this technique and would require multiple steps . As such, the separation of these subclasses is considered challenging.…”

“…In terms of quantitative analysis, this is commonly performed using the ELISA technique . However, the reagents used are also expensive and analysis is time consuming, frequently taking hours to complete . Other techniques that are available such as radial immunodiffusion and nephelometry, also tend to suffer from one or more of these disadvantages .…”