title: Study on the scale-up of human IgG3 purification using protein A affinity chromatography.
abstract: The purification of human IgG3 subclass out of IgG (Immunoglobulin-G) was studied using protein A-Sepharose affinity chromatography. The effect of operational parameters such as flow rate, ionic strength, pH and size of sample was investigated, and the process was scaled-up 10-fold. The use of 0.5 m NaCl in the loading buffer had a dramatic effect in the purity of IgG3 recovered in the flowthrough fraction (values in the order of 97% were consistently obtained). This was attributed to a more effective binding of IgG subclasses 1, 2 and 4 to protein A (well known classical mechanism based in Fc fragment) and in some extent to a decrease in the binding of subclass 3 to protein A by the alternative mechanism based in the Fab fragment. The increase in residence time also increased in a relevant way the purity of IgG3. This is attributed to an increased effectiveness of the mechanisms mentioned above. The recovery yields in the IgG3 rich fraction were in the range 21-32% and are possibly a consequence of binding to protein A by the alternative mechanism and also due to deactivation during processing.
authors.affiliation: Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal. jeamaral@skim.com
authors.forename: J
authors.initials: J
authors.lastname: Amaral
authors.forename: M
authors.initials: M
authors.lastname: Inganäs
authors.forename: J
authors.initials: J
authors.lastname: Cabral
authors.forename: D
authors.initials: D
authors.lastname: Prazeres
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000302:isolation & purification*; D009994:Osmolar Concentration; D012685:Sepharose / Q000031:analogs & derivatives*; D013205:Staphylococcal Protein A
chemical_list: D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; C042030:Staphylococcal protein A-sepharose; D012685:Sepharose