See also Antibody Purification Schemes and  Particular Types of Antibodies

Due to the significance of monoclonal antibody in medicine, there has been an increase in the number of purificaiton methods which also reflects the heterogeneity of this group of molecules and the various demands made on purity. The most common methods of purifying mabs, such as ammonium sulphate precipitation, protein A affinity chromatography and ion-exchange chromatography, result in a “co-purifying” of non-specific Ig Schuler (J. Chromatogr. 587(1), 61-70 (1991).

For the purification of an antibody by ion exchange, the antibody must have a charge opposite to that of the functional group attached to the ion exchange material. For example, antibodies, which generally have an overall positive charge in the buffer pH below its Pi, will bind well to cation exchange material, which contain negatively charged functional groups (WO 2010/048192, p. 31, lines 1-5). Because this interaction is ionic, binding must take place under low ionic conditions. Elution is achieved by increasing the ionic strenght to break up the ionic interaction, or by chaning the pH of the protein (US7825223).

Antibodies have been reported to have isoelectric points within the range 6-8 and, therefore, are ideal for purification by ion-exchange. Ion-exchange chromatography, while less selective than affinity, offers an opportunity for the isolation of immunoglobulins, either using anion-exchange at pH>pI or cation-excahnge at pH<pI (Denton”direct isolation of monoclonal antibodies from tissue culture supernantant using the cation-exchange cellulose Express-Ion S” J. chromatography A, 908 (2001) 223-234). Because antibodies have a more basic isoelectric point than the majority of other serum or contaminating proteins, ion exchange chromatograph (EIC) is useful in purifying antibodies regardless of isotype. The general strategy is to keep the pH below the isoelectric point for antibodies (proteins having a PI in bhte basic (range) will thus be positively charged under normal process conditions (pH below the PI of the protein)) so that they will not bind to an anion exchanger such as DEAE modified resin or, alternatively, to raise the pH above the pI where the antibodies will bind to the DEAE-groups. The opposite strategy works for cation exchangers. The bound antibodies are commonly eluted with a salt or pH gradient. Due to the fact that every antibody is unique and can vary in its pI, binding to an EIC resin needs to be explored and determined experimentally on an individual basis. The contaminant spectrum with which an antibody is associated on a cation exchanger is usually different from the spectrum on an anion exchanger. Even on the same exchanger, altering the pH may significantly change the profile of contaminants co-eluting with the antibody (Josic “Analytical and Preparative methods for purificaiton of antibodies” 2001). Due to their chemical and physical property, such as molecular weight and domain architecture, including secondary modifications, the downstream processing of immunoglobulins is very complicated (12/744089).

Challenges in Antibody Purification:

The challenge of defining a broadly applicable startegy for antibody purification is compounded by the chemical diversity of MAbs; they differ with respect to the number and distribution of their anionic, cationic, and hydrophobic residues. MAb concentrations vary significantly, as do the identity and proportion of contaminants in various growth medi (Gagnon, “A systematic approach to the purification of monoclonal antibodies” LCGC, 11(1), 1993). Due to the significant physicochemical differences that exist amoung mAbs, making a pre-defined purificaiton process for all mAbs is either impractical or results in a non-robust process (Shukla, J. Chromatogr. B 848 (2007) 28-39).