Elution by Change from Neutral to Acidic pH

Elution of the target molecule form the Protein A column generaly requires drastic conditions, a step gradient to 0.1M Glycine-HCL (pH<3.1) is typical. Elution at low pH contributes to virus inactivation, but threatens the yield of biologically active antibody (Schubert, J. Chromatography A, 1142 (2007) 106-113). 

(i) pH/Acids:  It has been shown that a conserved histidyl residue in the center of Protein A binding of IgGs faces a complementary histidyl residue on Protein A. These residues take on a positive charge at low pH, thus repelling each other and weakening the Protein A-IgG hydrophobic association. As a result, operating conditions for Protein A columns typically require the use of low pH conditions (typically between pH 3 and 4) for column elution (Shukla, J. Chromatography A, 1171 (2007) 22-28). 

For elution at pH <3, either citrate or acetate may be used.   The protein A column may typically be run in 4-10 cycles to purify a single batch. Since each cycle is only about 1 hour long, this cycling allows rapid throughput while reducing the cost of the column ((Fahrner, Biotechnol Genet Eng Rev, 2001, 18, 301-27 “Industrial purificaiton of pharmaceutical antibodies: development, operation, and validation of chromatography processes).

Cunningham (US 6800740) teaches purificaiton of MAbs from mouse ascites fluid by bidning to Protein A Sepahorse and elution with 0.1 M acetate (pH 3.0).

Fahrner (US 7485704 and US 2005/0038231) disloses a method of purifying a protein such as an antibody by protein A affinity chromatography and eluting with 0.1 M acetic acid.

Zhou (US2008/0132688) discloses passing a culture media containing Mab over protein A column and elution using a low pH buffer (for example, pH 3.4, 50-100 mm acetic acid).

(ii) polyols: Following the recovery of antibody bound to protein A with an acidic elution buffer solution, a stabilizing agent in the fomr of a polyol compound such as polyethlene glycol, polyvinyl pyrrolidone or ethylene glycol to suppress aggregation has been added (EP 11568710).

Elution by Adding Additives

(i)amino acid addition: 

–Arginine: Yumioka (EP 1568710; see also US 2005/0176109 and US 2012/0142901) teaches a method of antibody purification with Protein A affinity by contacting the antibody with a buffer solution adjusted to pH 4.0 to 5.0 comprising arginine and/or a derivative used to desorb/elute the antibody from the column. Ejima (Analytical Biochemistry 34, 2005, 250-257) also disclose protein A affinity chromatography is enchanced significantly by using arginine as an eluent. Arakawa (Protein Expression and Purification 36 (2004) 244-248 also discloses that the recovery of antiboides was greatly increased with 0.5M arginine and more so with 2M arginie compared to the conventional eluent citrate.

(ii) Mutants of Protein A:  See outline (e.g., temperature sensitive Protein A mutatns).