The price of Protein A affinity chromatography resins is many times the cost of non-affinity supports. Accordingly Protein A resins are typically recyled by treatment with strong chaotropic solutions (urea and guanidinde hydrochloride) or strongly acidic solutions (such as acetic acid) or a combination thereof. Johansson (US2008/0230478)

Particular Agents used for Protein A Regneration

Equilibration buffer: The most commonly used cleaning is a simple wash with buffer, such as the equilibration buffer. Johansson (US2008/0230478)

Equilibration buffers include phosphate and carbonate buffers. Ladiwala (US 13/543650)

Neutralization agents: include phosphate buffers, weak acids and weak bases.

Acid treatment: Washing with an equilibrium buffer can only be used to restore the Protein A matrix a limited number of times. For a more efficient cleaning, treatments with acid and/or base are frequently used, each removing acid and base-sensitive contaminants respectively. Johansson (US2008/0230478)

Acidic solutions include phosphoric and acidic acid. Ladiwala (US 13/543650)

Alkaline protocol (NaOH) (Cleaning In Place (CIP)): In order to even more efficiently restor an Affinity A matrix, an alkaline protocol known as CIP is commonly used with many matrices. The standard CIP involves treatment with 1 M NaOH, pH 14. This harsh treatment will efficiently remove undesired fouling asuch as by protein aggregates and the like, but may on the other hand impair some chromatography matrices. For example, many affintiy matrices, wehre the ligands are proteins cannot withstand standard CIP. Protein A ligands need to be cleaned under milder conditions than conventional CIP in order to maintain selectivity and binding capacity. Thus such a sensitive matrix may be cleaned with standard CIP, if a reduced performance is acceptable. Johansson (US2008/0230478) However, the most extensively used cleaning and sanitising agent is NaOH in a concentration ranging from 0.1 up to 1 M, depending on the degree and nature of contamination. (Ander, 14/385336). 

Chaotropic agents: include guanidine HCl, urea and guanididne acetate. Ladiwala (US 13/543650)

Protein A media can be re-used after cleaning with 6M urea or 6 M guanidine hydrocholoride, which are known as milder cleaning buffers than sodium hydroxide. However, urea is relatively costly and due to its fertilising effect, it cannot be readily disposed of without taking certain precautions. Johansson (US2008/0230478)

Reducing agents: include thioglycerol, 1-4, dithiothreitol and 2-mercaptoethanol. Ladiwala (US 13/543650)

As the skilled person in the field will recognize, adding a reducing agent in a chromatographic process may entail the risk of retaining reducing agent in the matrix, which could potentially harm or contaminate the target molecule. By performing at least one of acidic and alkaline regeneration subsequent to the addition of reducing agent, such risk is minimized. Johansson (US2008/0230478)

Organic soolvents:

Braunger (US 2005/0056592) discloses reeenration of absorbent matrices such as Protein A where the regeneration solution includes an organix solvent such as n- or isoproanol. water-miscile alchols such as methanol, ethanol, t-butanol and secondary butanol, isobutanol.  In one ebmodiment, both an acid solution and an alkaline solution are used consecutively in one, more or all the cycles, preferably with the acid solution preceding the alkaline solution. The organic solvent is prefereable always included in the first of them. 

Chloridne dioxide:

Hamilton (US 2007/0093399) disclsoes that chlorine dioxide is an effective method of cleaning and regenerating chromatography media. 

Regeneration Schemes

Acid solution – Aklaine solution:

Bian (US2016/0193633) discloses methods for cleaning a Protein A chromatography column using both acidic (e.g., H3PO4) and alkaline (eg.g, NaOH) solutions in an alternating manner. 

Braunger (US2005/0056592) discloses a regeneration scheme for adsorbent matrices using an organic sovlent having a pH less than 4 (the acid components as part of the buffer scheme include carboxylic acids such as acetic acid, citric acid, malic acid) and then an alkaline solution with a pH greater than 10 but less than 13. The scheme that is the combination of an acidic solution which contains an organic solvent, preferably isoprobanol or n-propanol, and a second regeneration step with an alkaline solution solution, preferably 5-40 mM sodium hydroxide results in a surprisnly effective regeneration of the matrix with no blockage of the column after more than 100 cycles. 

Mahajan (US 14/479092, published as 2015/0093800 and US 15/920237, published as US 2019/0054396) discloses a method to clean a chromatogrpahy material for reuse by passing two or more material volues of elution buffer comprising about 0.15 M acetic acid at about pH 2.9, statically holding the material in elution buffer for about 10-30 minutes, passing two or more material volumes of elution buffer and then passing about two or more material volumes of regeenration buffer comrpsing about 0.1 N NaOH at about pH 13. 

Chaotropic agent + reducing agent: 

Madani (US6,972,327) teaches a method for regenerating a Protein A chromatography resin by washing the resin with a chaotropic solution such as guanidium HCL containing a reducing agent which is a source of free thils such as glutathione, dithiothreitol (DTT), 2-mercaptoethanol, dithionitrobenzoate (DTNB) and cysteine.

Ladiwaia (US13/543650) also teaches a method for regenerating an antibody binding resin by conducting two separate steps; a first wtep of washing the resin with a reducing solution selected from the group consisting of thioglycerol, 1-4,-dithiothreitol and 2-mercaptoethanol and a second step of washing with a chaotropic solution selected from the group consisting of guanidine HCL, urea and guanidine acetate.

-(acid solution) -(neutralization) -Reducing agent -(equilibration buffer) – chaotropic agent :

Ladiwala (US 13/543650) discloses a method for regenerating chromatography resins which includes a first step of washing the resin with a reducing solution having a reducing agent such as thioglycerol, 1-4-dithiothreitol or 20mercaptoethanol or thioglycerol followed by a second step of washing with a chaotropic solution which includes a chaotropic agent such as guanidine-HCL, urea or guanidine acetate.

Reducing agent -(acid solution) (–alkaline solution) – alkaline agent  -equilibrium: 

Johansson (US 2008/0230478) discloses regeernation of a seapration matrix by reducing regenration by contacting the matrix with a reducing agent, alkaline regeneration and equilibration of the matrix, wherein the steps can be performed in any order. In one embodiment, the process includes acidic regeenration by contacting the matrix with an acidic solution at any time after elution but before quilibration. In a specific embodiment, the acidic regeneration is carried out after the reducing regeneration. In one embodiment, acidic and alkaline regeneration are carried out subsequent to the reducing regeneration.