See also “Protein A – hydroxyapatite” under “protein A” and “chromatography”

Purification schemes typically include an affinity purification step, such as Protein A purificaiton in the case of antibodies. Despite a high binding affinity of Protein A chromatography for antibodies and ability to remove as much as 99.5% of impurities, it is an expensive purificaiton step on a commercial scale. Even if Protein A affinity chromatography is used, adequate purity is often not achieved unless several purificaiton steps are combined, thereby further increasing cost and reducing product yield.

I. Protein A/G Affinity Chromatography: See outline

II. Non Protein A/G Affinity Purification Schemes: 

Despite the fact that affinity chromatography is often employed as a capture step for antibody purification, the high cost and instability of affinity media as led to schemes using fewer steps and without the need for a costly affinity step.

A. Ion Exchange Chromatography: See outline

B. Mixed-Mode (Multimodal) Chromatography:

–Hydroxyapatite Chromatography: See outline

C. Filtration Methods: See outline

D. High Performance Liquid Chromatography (HPLC): Danielsson “”one-step purification of monoclonal IgG cantibodies from mouse ascities, J. Immunological methods, 115 (1988) 79-88 disclose purification of monoclonal antibodies in one step from mouse ascites by 4 different HPLC adsorption techniques; anion exchange (Mono Q), cation exchange (Mono S), chromatofocusing (Mono P) and hydrophobic interaction chromatography. They propose using cation exchange as a first choice for antibodies with high isoelectric points (>7.2) and hydrophobic interaction chromatography as a first choice when isoelectric point is below 7.2

E. Precipitation: See outline

F. Immobilized metal affinity chromatography (IMAC): can purify IgG molecuels due to the affinity binding of histidyl residues in the antibody molecules to a chelated nickel support. Mammalian IgGs ahve a highly conserved histidyl cluster at the junctures of the CH2 and CH3 domains. Under alkaline conditions, nickel loaded iminodiacetic acid (Ni-IDA) captures IgG through the histidyl residues electively and effectively. (Josic, Methods for purificaiton of antibodies, Food technol. biotechnol. 39 (3) 215-226 (2001).