Operating Conditions for the Binding/washing/elution

Binding:

(i) pH: It is known that binding of mouse IgG to protein A-Sepharose is pH sensitive and that optimum adsorption occurs using 0.1M sodium phosphate, pH 8.0 buffer (Ngo, 4801687). It has been known for some time that the binding of murine IgG1 to protein A is dependent on the pH. In contrast to the other murine IgG subclasses, complete binding cannot be achieved if the pH is less than 8.0. Recommendations have been made that binding is performed at pH values considerably higher than 8.0. Schuler (J. Chromatogr. 587(1), 61-70 (1991), however, did not find that raising the pH value from 8.2 to 8.7 gave any imporvement in yield at the saline concentrations and temperatures used in that stufy.

(ii) sodium chloride + glycine/ phosphate or Tris: Buffers with a very high concentration of saline are often recommended to strengthen the binding between protein and murine IgG, in particular, IgG1. Examples tof these concentrations are3M sodium chloride plus 1.5 M glycine, 2 M NaCL + 2 M glycine, 0.5 M phosphate or 1 M Tris. Improved binding is achieved by the intensiifcation of hydrophobic interaction (Schuler (J. Chromatogr. 587(1), 61-70 (1991).

Ngo (4801687) teaches a process for the purification of antibodies which includes the step of mixing the meidum containing antibodies with a buffer solution having a pH in the range of about ph7.5 to pH 10 which contains a hydrophobic interaction promoting salt in a concentration of about 0.6M to 1.75M and then containing the resulting buffered medium with protein A to adsorb. Any buffer may be used to provide the desired pH. For example, glycine buffer, borate buffer or trsi buffer can be used in the range of about 0.01M to 0.25M.

(iii) Temperature: has the most significant effect on the strenght of binding which may occur by weakening hydrophobic interactions. With a weakening of the intramolcular hydrophobic bonds, slight changes may be induced in conformation in either one or both binding partners which resutls in an overall increase in affinity. It is thus possible to effectively purify murine IgG1 mabs from culture supernatants using protein A affinity chromatography with a colled column Schuler (J. Chromatogr. 587(1), 61-70 (1991).

Wash: 

–Aliphatic carboxylate (e.g., sodium caprylate, sodium decanoate or sodium dodecanoate):

Goklen (US 14/775,868US2016/0024146; see also US Patent Application No: 15/931,062, published as US 20200277330) discloses a method for purifying a protein using Protein A chromatogrpahy by absorbing the protein to the Protein A and removing a contaminant using a wash buffer that includes an aliphatic carboxylate such as sodium caprylate, sodium decanoate or sodium dodecanoate. In one embodiment the buffer further includes sodium acetate. 

–Arginine:

Sun (US 20080064860 and 20080064861; see also WO/2008/030120) disclose washing affinity chromatography colun with a wash buffer that contains arginine). 

Frauenschuh (US 13/516960 and US2012/0283416, now US 9,505,803) disclose a wash solution for affinity chromatography using both arginine and a nonbuffering salt, such as a halogen salt (e.g., sodium, potassium, calcium or magnesium chloride). . Preferably the wash solution is at high pH. 

Eckermann (US13/522030, published as US 9,284,347) disclose a wash buffer for protein A chromatography which contains 1) arginine, 2) sodium chloride 3) an alcohol such as isopropanol, n-propanol and ethanol and 4) polyvinylpyrrolidone and/or a detergent.

Morii (EP0333474) discloses removal of endotoxin contaminants from an aqueous solution of a valuable protein such as an antibody using affinity chromatography (does not disclosve Protein A in particular) which includes a wash with pH in the range of 4-11 and an amino compound such as arginine at a concentration 1-500 mM.

–Chaotropic agents/ detergents (e.g., Urea) : are protein denaturants that dissociate hydrogen bonds and affect the termtiary sturcture of the proteins. Shukla (WO 2007/109163)

Shukla (WO 2007/109163) discloses a method for purifying a protein using Protein A chromatography taht includes a wash buffer that includes haotropic agents, hydrophobic modifiers and agents that reduce electrostatic interactions for removing host cell contaminants. Examples include urea, guanidinium hydrochloride and sodium thiosulfate. Getergents are examples of hydrophobic modifies that act by competing for hydrophobic sites and inlcude noionic surfactants such as Polysorbate 20 (Tween 20) and Polysorbate 80 (Tween 8), Triton, SDS. sodium larel sufate. 

–Salts: 

Protein A chromaotgraphy wash buffers containing salt (such as sodium chloride) alone or in combination with either a detergent (e.g., tween 20), a solvent (e.g., hexylene glycol) or a polymer (e.g., polyethylene glycol) have been described (UBreece, 6,870,034). 

One way to decrease the amount of host cell proteins in the elution pool when using Prosep media is by using an intermediate wash with tegramethylammonium chloride(TMAC) (Fahrner, Biotech. Genet. Eng. Rev. 18, 2001, p. 311, 2nd ¶). 

–No Salts: (only Tris base, acetic acid & sodium acetate)

Goklen (WO 2012/135415) discloses equilibrating a Protein A with a buffer that includes 55 mM tris base, 45 mM acetic acid at about pH 7.5, (b) adsorbing the protein from the contaminated solution to the Protein A and (c) removing contaminants by washing with a wash buffer that includes 55 mM Tris Base, 45 mM acetic acid, 300 mM sodium acetate at about pH 7.5 and recovering the protein with an elution buffer that includes 1.8 mM sodium acetate, 28.2 mM aetic acid, at about pH 3.6 wherein all the buffers are made without the addition of NaCl. 

Elution:  See outline

Operating Conditions for the Entire Procedure

 Ligand Density:

Berg (WO2006/065208) discloses a matrix to which antibody binding protein ligands such as Protein A, G and/or L have been immobilised. In a preferred embodiment, the ligands comprise a monomer, dimer or multimer of Protein A domains (e.g., one or more of Domain A, B, C, D and E domains or a dimer or multimer of Protein Z). The ligands may be immobilised using well known methods such as epoxi coupling. In a specific emobdiment, the ligand density is in the range of 5-10 mg/ml. 

 Temperature/Protease inhibitors: 

Fahrner (US 7485704 and US 2005/0038231) disloses a method for reducing leaching of protein A during protein A affinity chromatography by reducing temperature or pH or by adding one or more protease inhibitors to a composition that is subjected to protein A affinity chromatography. In one embodiment the termpature is in the range form about 3C -20C.

 Nielsen disclloses a method for reducing leaching of protein A by reducing temperature or pH or by adding protease inhibitor(s) to a ocmposition that is subjected to protein A affinity chromatography (WO 2005/016968).