WO2010066676A1

WO2010066676A1 - Method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column

Info

Publication number: WO2010066676A1
Application number: PCT/EP2009/066525
Authority: WO
Inventors: Robert John Noel; Allan Otto Fog Lihme
Original assignee: Upfront Chromatography A/S
Priority date: 2008-12-09
Filing date: 2009-12-07
Publication date: 2010-06-17

Abstract

A method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column comprising the step of contacting said column with a cleaning solution at an elevated temperature with substantial retainment of activity of covalently immobilised adsorption ligands. Said method provides efficient removal of unwanted material and allows repeated use of said column while maintaining stability thereof.

Description

TITLE OF INVENTION

METHOD OF REMOVAL OF UNBOUND, ENTRAINED AND/OR NON-SPECIFICALLY ADSORBED MATERIAL FROM AN AFFINITY CHROMATOGRAPHY COLUMN

FIELD OF THE INVENTION

The present invention relates to a method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatrography column. More particularly the present invention relates to a method of removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed adsorption chromatography column with substantial retainment of activity of covalently immobilised adsorption ligands, e.g. protein A.

BACKGROUND OF THE INVENTION

Adsorbents for the capture of target molecules, such as biomolecules consisting of immobilised large molecule affinity ligands, such as antibodies, immunoglobulin-binding proteins, antigens, proteins, glycoproteins, enzymes etc. are used because they represent a high degree of binding specificity towards target molecules.

Economical manufacture of biomolecules such as therapeutic antibodies, enzymes, receptor blocking agents etc. demands the repeated use of such adsorbents over many production batches to lower the impact of the high purchase cost. Such adsorbents are more liable to fouling, degradation and limited re-use because they cannot be cleaned with aggressive cleaning agents such as IM NaOH.

The aim of an adsorbent cleaning regime is to remove all biomolecules other than those covalently immobilised to the adsorbent in between multiple cycles of adsorption. These includes mammalian cells, bacteria, fragments of cellular organisms, proteins, nucleic acids, lipids, etc. derived from cellular organisms, which can either degrade the immobilised adsorption ligands or block the ligands from interacting with the solution or physically block pores in the adsorbent so that target molecules cannot reach the immobilised adsorption ligands. Cleaning of the adsorbent sets out to remove contaminants and allows consistent operation of the adsorbent. A further aim of an adsorbent cleaning regime is to ensure that no impurities are carried over from one adsorption cycle to the next adsorption cycle and even more importantly to ensure that no impurities are carried over from the manufacture of one batch of product to the next batch of product.

The most commonly used prior art cleaning method is a simple wash with buffer at room temperature, such as the equilibration buffer. Such washing can only be used to restore the 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. In order to more efficiently restore the matrix, an alkaline protocol known as Cleaning In Place (CIP) is commonly used with many matrices. The standard CIP involves treatment of the matrix with IM NaOH, pH 14. Such harsh treatment will efficiently remove undesired fouling such as by protein aggregates and the like, but many affinity matrices, wherein the ligands are proteins or proteinaceous, cannot withstand standard CIP, at least not while maintaining their integrity and activity. For example, one of the most commonly used affinity chromatography matrices for purification of antibodies comprises Protein A ligands, but such matrices must be cleaned under milder conditions than conventional CIP in order to maintain selectivity and binding capacity.

US7052609 discloses a regeneration method for hydrolysis sensitive adsorbents in chromatography processes, which involves contacting adsorbent matrix with aqueous regeneration solution(s) containing water-miscible organic solvent and having specific pH.

US20080230478A1 relates to a process of regenerating a separation matrix, such as a chromatography matrix, comprising adsorption of at least one target molecule by contacting a mobile phase comprising at target molecule(s) with a matrix; removal of unbound material by washing the matrix; elution of target molecule(s) by contacting the matrix with an eluent; reducing regeneration by contacting said matrix with a reducing agent; alkaline regeneration by contacting the matrix with an alkaline solution; and equilibration of the matrix.

US6972327 discloses a method for regenerating a chromatography resin, which comprises eluting from a protein A or G chromatography resin an antibody or Fc fusion protein that has been bound to it and regenerating the chromatography resin by washing with a chaotropic solution containing a reducing agent

Brorson et al (Kurt Brorson, Janice Brown, Elizabeth Hamilton, Kathryn E. Stein in Journal of Chromatography A, 989 (2003) 155-163: Identification of protein A media performance attributes that can be monitored as surrogates for retrovirus clearance during extended re-use") describes how Protein A media can be reused after cleaning with 6M urea or 6 M guanidine hydrochloride, which are known as milder cleaning agents than sodium hydroxide. Column performance was said to be stable even after more than 300 cycles. However, the use of urea involves certain drawbacks, firstly due to its cost and secondly, due to its fertilising effect, it cannot be readily disposed of without taking certain precautions.

Thus, there is still a need in the art of alternative cleaning protocols for chromatography matrices, especially for use with more sensitive materials such as proteinaceous or protein-based ligands.

Especially expanded bed adsorption columns need improved and efficient cleaning methods that has minimal deleterious effect on the adsorption ligand because typically this type of column is utilised for the isolation of target biomolecules from crude raw materials that has not been clarified by prior filtration and/centrifugation. Thus expanded bed columns are exposed and challenged by significantly higher concentrations of cell debris and other insoluble impurities that may be entrained in the bed and/or non-specifically adsorbed to the adsorbent. Separation processes that operate at industrial scale routinely use increased temperatures to clean adsorbents for multiple operational cycles. Increased temperatures have a pasteurising effect on the adsorbent, kinetically accelerate cleaning action and also use the higher solution entropy as an additional cleaning effect.

The use of cleaning solutions at high temperatures have not been utilised with large molecules affinity adsorbents in commercial operations for fear of molecular instability and other operational problems. The addition of high temperature solutions to fixed or packed beds of adsorbents presents operational problems, not least degassing of solutions and the introduction of air bubbles directly into the fixed bed which degrades performance and necessitates adsorbent re-packing.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide a method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column which provides efficient cleaning and allows repeated use of said column.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that efficient removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column may be provided by the use of a cleaning solution at elevated temperatures for the removal thereof while still maintaining an acceptable stability of said column.

So, in a first aspect the present invention relates to a method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column comprising the step of contacting said column with a cleaning solution at an elevated temperature with substantial retainment of activity of covalently immobilised adsorption ligands.

LEGENDS TO THE FIGURES

Fig. 1 shows the relative IgG binding capacity of a protein A adsorbent treated with demineralised water as the cleaning agent at pH 6.4 and at 50 ⁰C, 60 ⁰C, 70 ⁰C and 80 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 2 shows the relative IgG binding capacity of a protein A adsorbent treated with 0.1 % and 1 % sodium dodecyl sulphate in demineralised water as the cleaning agent at pH 7.2 and at 50 ⁰C, 60 ⁰C and 80 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 3 shows the relative IgG binding capacity of a protein A adsorbent treated with 10 mM dithiothreitol in demineralised water as the cleaning agent at pH 6.5 and at 50 ⁰C and 60 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 4 shows the relative IgG binding capacity of a protein A adsorbent treated with 1 % sodium lauroyl sulphate in demineralised water as the cleaning agent at pH 6.4 and at 50 ⁰C. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 5 shows the relative IgG binding capacity of a protein A adsorbent treated with 20 % 1,2 propanediol in demineralised water as the cleaning agent at pH 7.4 and at 50 ⁰C and 80 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 6 shows the relative IgG binding capacity of a protein A adsorbent treated with 60 % 1,2 propanediol (MPG) in demineralised water as the cleaning agent at pH 7.5 and at 70 ⁰C. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity. Fig. 7 shows the relative IgG binding capacity of a protein A adsorbent treated with 1 M sodium chloride in demineralised water as the cleaning agent at pH 7.5 and at 50 ⁰C and 60 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 8 shows the relative IgG binding capacity of a protein A adsorbent treated with 1 M and 3 M magnesium chloride in demineralised water as the cleaning agent at pH 6.8 at room temperature and at 50 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

Fig. 9 shows the relative IgG binding capacity of a protein A adsorbent treated with 0.1 M sodium acetate in demineralised water as the cleaning agent at pH 4.0 and pH 5.0 and at 50 ⁰C and 60 ⁰C respectively. Untreated protein A adsorbent is defined as the reference having 100 % binding capacity.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

In the present context an "affinity chromatography column" refers to a chromatography column which is based on specific interactions between a target (bio)molecule and a (bio)specific ligand in a principle of lock-key recognition. Thus, the target and ligand will constitute an affinity pair, such as antigen/antibody, enzyme/receptor, protein A/immunoglobulin etc.

In the present context an "expanded bed adsorption chromatography column" refers to an affinity chromatography column in which the solid phase is in a fluidised state during operation of the column. In the present context the term "elevated temperature" refers to a temperature above room temperature, such as a temperature of at least 30 ⁰C.

In the present context the term "adsorption ligand" refers to any ligand which is covalently immobilised to the solid phase of an affinity chromatography column and is capable of binding a target molecule. Exemplary adsorption ligands include antibodies, antigens, immunoglobulin-binding proteins, proteins, peptides, glycoproteins, enzymes etc.

In the present context the term "target molecule" or "target biomolecule",said terms being used interchangeably, refers to any (bio)molecule that adsorbs to the affinity chromatography column via the adsorption ligand and embraces compounds and cells as well as actual molecules. Exemplary target molecules are proteins, enzymes, nucleic acids, plasmids, immunoglobulins, peptides, hormones, such as monoclonal antibodies and fragments hereof.

In the present context the term "unbound, entrained and/or non-specifically adsorbed material" refers to any unwanted material from a production batch of a wanted target (bio)molecule, such as mammalian cells, bacteria, fragments of cellular organisms, proteins, nucleic acids, lipids, etc. derived from cellular organisms, which can either degrade the immobilised adsorption ligands, block the ligands from interacting with the solution or physically block pores in the adsorbent so that target molecules cannot reach the immobilised adsorption ligands or pose a risk of cross-contamination between successive batches of produced target biomolecule reusing the same batch of adsorbent.

In the present context the term "substantial retainment of activity of covalently immobilised biomolecules" refers in the present context to the fact that the activity of adsorption ligands immobilised to the adsorbent before treatment thereof according to the invention is not reduced significantly by said treatment. By the term "substantial retainment of activity" in the present context is meant that the activity of the immobilised adsorption ligand is not reduced by more than 25 % within a period of 24 hours of continuous treatment, preferably is not reduced by more than 20 % within a period of 24 hours of continuous treatment , such as not more than 15 % within a period of 24 hours of continuous treatment, preferably is not reduced by more than 25 % within a period of 50 hours of continuous treatment, such as not more than 25 % within a period of 100 hours of continuous treatment.

In practice the treatment according to the invention will be applied to the adsorbent in between each adsorption cycle or after a predetermined number of adsorption cycles in order to secure continuously efficient and safe performance of the adsorption process. As a minimum, the treatment according to the invention will be applied in between the adsorption of different batches of the target biomolecule to be produced. The treatment may comprise a sequence of steps using different cleaning solutions and/or different temperatures. Typically each treatment will be performed for a period of time ranging from about 10 minutes to 4 hours. In order to establish the stability of the immobilised adsorption ligand towards a preferred treatment according to the invention and using a specific cleaning solution, prior to implementation of the treatment in an industrial production scenario, the adsorbent may be continuously treated at laboratory scale for prolonged periods of time (e.g. 24 hours or hundreds of hours) followed by measurement of the activity of the adsorption ligand.

In the present context the term "activity" of the immobilised adsorption ligand refers to the intended function of the immobilised adsorption ligand for adsorption of a target molecule when applied in an affinity chromatography process. A typical measure of the "activity" in this context is the dynamic capacity and/or the static binding capacity for the adsorption ligand, typically measured in milligram target biomolecule bound per millilitre adsorbent under defined and standardised conditions.

In the present context the term "cleaning solution" refers to a liquid composition which when passed through an affinity chromatography column is capable of removing any unbound, entrained and/or non-specifically adsorbed material therefrom. In the present context the term "cleaning agent" refers to an agent which when added to the cleaning solution has an additional cleaning effect relative to pure water. Typical cleaning agents belong to the group of surface active agents such as cationic, anionic, zwitterionic and non-ionic surfactants, organic solvents such as alcohols, inorganic or organic salts, chaotropic reagents such as chaotropic salts such as magnesium chloride, potassium iodide, potassium thiocyanate, urea, and guanidinium. hydrochloride, reducing agents such as dithiothreitol and mercaptoethanol, sodium sulfite; oxidising reagents such as hydrogen peroxide and chlorine - or any combination of two or more of the above.

In the present context the term "stabilising agent" refers to an agent which when added to the cleaning solution exerts a stabilising effect on the affinity chromatography column, i.e. exerts a stabilising effect on the interaction between target (bio)molecule and adsorption ligand.

Specific embodiments of the invention

In an embodiment of the method according to the invention the elevated temperature is a temperature of at least 35 ⁰C.

In an embodiment of the method according to the invention the elevated temperature is a temperature of at least 40 ⁰C.

In an embodiment of the method according to the invention the elevated temperature is a temperature of at least 50 ⁰C.

In an embodiment of the method according to the invention the elevated temperature is a temperature of at least 60 ⁰C.

In an embodiment of the method according to the invention the elevated temperature is a temperature of at least 70 ⁰C.

Thus the temperature chosen in each individual case will vary depending on a number of factors, such as the amount and nature of contaminants to be removed, the nature of the target molecules attached to the adsorbent, the specific character of the adsorbent in question etc. and will most often be a compromise between on the one hand efficient and fast cleaning and on the other hand the need for taking due account of the sensitivity of the target (bio)molecule-adsorption ligand affinity.

In an embodiment of the invention the affinity chromatography column is an expanded bed column.

Expanded bed adsorption operations allow the free passage of air bubbles through the adsorption particles because they are not fixed or packed together. It is therefore possible to rapidly change solution temperatures without experiencing operational problems experienced with air bubbles previously seen with other affinity chromatography columns.

In an embodiment of the invention the adsorption ligand is a protein or a proteinaceous material.

In an embodiment of the invention the adsorption ligand is Protein A, Protein G or an antibody, preferably Protein A.

In an embodiment of the method according to the invention the cleaning solution is water.

Thus it has turned out that an efficient removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed adsorption chromatography column with substantial retainment of activity of covalently immobilised biomolecules (adsorption ligands) may be obtained by the use of an aqueous solution, such as water at an elevated temperature.

In an embodiment of the invention the cleaning solution comprises one or more cleaning agents. In an embodiment of the invention said cleaning agents are selected from the group consisting of surface active agents such as cationic, anionic, zwitterionic and non-ionic surfactants, organic solvents such as alcohols, inorganic or organic salts, chaotropic reagents such as chaotropic salts, urea, and guanidinium. hydrochloride, reducing agents, oxidising reagents or any combination of two or more of the above.

As illustrative, non-limiting examples of anionic surfactants may be mentioned sodium dodecyl sulphate (SDS), Na-N-lauroylsarcosinate, Chenodeoxycholic acid, Cholic acid, Dehydrocholic acid, Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin, Digitoxigenin, N,N-Dimethyldodecylamine N-oxide Docusate sodium salt, Glycochenodeoxycholic acid sodium salt, Glycocholic acid hydrate, Glycocholic acid sodium salt, Glycodeoxycholic acid, Glycodeoxycholic acid sodium salt, Glycolithocholic acid 3-sulfate disodium salt, Glycolithocholic acid ethyl ester, Lugol Niaproof 4, Triton QS-15 QS-15 Triton QS-44 solution, 1- Octanesulfonic acid sodium salt, Sodium 1-butanesulfonate, Sodium 1- decanesulfonate, Sodium 1-dodecanesulfonate, Sodium 1-heptanesulfonate, Sodium 1-heptanesulfonate, Sodium 1-nonanesulfonate, Sodium 1- propanesulfonate monohydrate, Sodium 2-bromoethanesulfonate, Sodium hexanesulfonate, Sodium octyl sulfate, Sodium pentanesulfonate, Sodium taurocholate Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodium salt , Taurodeoxycholic acid sodium salt, Taurohyodeoxycholic acid sodium salt, Taurolithocholic acid 3-sulfate disodium salt, Tauroursodeoxycholic acid sodium salt, Triton X-200 Ursodeoxycholic acid.

As illustrative, non-limiting examples of cationic surfactants may be mentioned :

Alkyltrimethylammonium bromide, Benzalkonium chloride,

Benzyldimethylhexadecylammonium chloride,

Benzyldimethyltetradecylammonium chloride, Benzyldodecyldimethylammonium bromide, Benzyltrimethylammonium tetrachloroiodate,

Dimethyldioctadecylammonium bromide, Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide, Dodecyltrimethylammonium bromide, Ethylhexadecyldimethylammonium bromide, Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammonium bromide, N,Nφ,Nφ-Polyoxyethylene(10)-N-tallow-l,3-diaminopropane, Thonzonium bromide, Trimethyl(tetradecyl)ammonium bromide.

As illustrative, non-limiting examples of zwitterionic surfactants may be mentioned

CHAPS, CHAPSO, 3-(Decyldimethylammonio)propanesulfonate inner salt 3-(Dodecyldimethylammonio)propanesulfonate inner salt, 3-(Dodecyldimethylammonio)propanesulfonate inner salt, 3-(N, N- Dimethyl myristylammonio)propanesulfonate, 3-(N,N-Dimethyloctadecylammonio)propanesulfonate,

3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt, 3-(N, N- Dimethyl palmitylammonio)propanesulfonate.

As illustrative, non-limiting examples of nonionic surfactants may be mentioned

Bis(polyethylene glycol bis[imidazoyl carbonyl]), Brij^® 35, Brij^® 56, Brij^® 72, Brij^® 76, Brij^® 92, Brij^® 97, Brij^® 58P, Cremophor^® EL, Decaethylene glycol monododecyl ether,

N-Decanoyl-N-methylglucamine, n-Decyl -D-glucopyranoside, Decyl -D- maltopyranoside, n-Dodecanoyl-N-methylglucamide, n-Dodecyl -D-maltoside, Heptaethylene glycol monodecyl ether, Heptaethylene glycol monododecyl ether, Heptaethylene glycol monotetradecyl ether, n-Hexadecyl -D-maltoside,

Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecyl ether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycol monotetradecyl ether, Igepal CA-630,

Methyl-6-0-(N-heptylcarbamoyl)- -D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-Nonanoyl-N-methylglucamine, N-Nonanoyl-N- methylglucamine,

Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether

Octaethylene glycol monotetradecyl ether, Octyl- -D-glucopyranoside, Pentaethylene glycol monodecyl ether, Pentaethylene glycol monododecyl ether Pentaethylene glycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,

Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctyl ether

Polyethylene glycol diglycidyl ether, Polyethylene glycol ether, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate, Polyoxyethylene 20 isohexadecyl ether,

Polyoxyethylene 20 oleyl ether, Polyoxyethylene 40 stearate,

Polyoxyethylene 50 stearate, Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl), Polyoxyethylene 25 propylene glycol stearate, Span^® 20, Span^® 40, Span^® 60, Span^® 65, Span^® 80, Span^® 85, Tergitol, Type 15-S- 12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5, Tergitol, Type 15-S-7,

Tergitol, Type 15-S-9, Tergitol, Type NP-IO, Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7, Tergitol, Type NP-9, Tergitol, Type TMN-10, Tergitol, Type TMN-6, Tetradecyl- -D-maltoside, Tetraethylene glycol monodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethylene glycol monotetradecyl ether,

Triethylene glycol monodecyl ether, Triethylene glycol monododecyl ether, Triethylene glycol monohexadecyl ether, Triethylene glycol monooctyl ether, Triethylene glycol monotetradecyl ether, Triton CF-21, Triton CF-32, Triton DF- 12, Triton DF-16, Triton GR-5M, Triton X-IOO, Triton X-102, Triton X-15, Triton X-151, Triton X-207, Triton^® X-114,

Triton^® X-165 solution, Triton^® X-405 solution, TWEEN^® 20, TWEEN^® 21, TWEEN^® 40,

TWEEN^® 60, TWEEN^® 61, TWEEN^® 65, TWEEN^® 80, TWEEN^® 81, TWEEN^® 85, Tyloxapol. As illustrative, non-limiting examples of alcohols may be mentioned ethanol, propanol, 1,2-propanediol (MPG), glycerol.

As illustrative, non-limiting examples of inorganic and organic salts may be mentioned alkaline metal and alkaline earth metal salts, such as NaCI, Na₂CO₃, Na₂SO₄, , K₃PO₄

As illustrative, non-limiting examples of chaotropic agents may be mentioned MgCI₂. potassium iodide, sodium thiocyanate, urea, guanidine hydrochloride.

As illustrative, non-limiting examples of reducing agents may be mentioned dithiothreitol and mercaptoethanol.

As illustrative, non-limiting examples of oxidising agents may be mentioned hydrogen peroxide and chlorine.

The amount of each cleaning agent added varies widely depending on the particular agent used. However, amounts of e.g. surfactant in the range 0.01- 5% by weight, such as 0.05-3 % by weight, such as 0.1-1 % by weight may be used. For other cleaning agents, such as alcohols, amounts up to 60-70% of the cleaning solution may be employed, such as 40-60 % of 1,2-propanediol.

In an embodiment of the method according to the invention the aqueous solution comprises one or more stabilising agents. Thus in order to stabilise the affinity chromatography column against unwanted inactivation of the ligand and thus loss of binding capacity during cleaning of the column, in an embodiment of the invention the use of one or more stabilising agents is foreseen.

In an embodiment of the invention said stabilising agents are selected from the group consisting of inorganic and organic salts, organic and inorganic acids and derivatives thereof, surfactants, alcohols, ethers, esters, aldehydes, ketones, sugars, polymers, polysaccharides. An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with water as a cleaning solution at a temperature of 50 ⁰C.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS in water at a temperature of 50 ⁰C.

In an embodiment of the method according to the invention a solution comprising 0.05-5 %, such as 0.1 % to 1 % SDS in water at a temperature of 50 ⁰C is employed as cleaning agent.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising sodium lauroyl sarcosinate in water at a temperature of 50 ⁰C.

In an embodiment of the method according to the invention a solution comprising 0.05-5 %, such as 0.1 % to 1 % sodium lauroyl sarcosinate in water at a temperature of 50 ⁰C is employed as cleaning agent.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising magnesium chloride in water at a temperature of 50 ⁰C.

In an embodiment of the method according to the invention a solution comprising 0.5 M to 5 M, such as 1 M to 3 M magnesium chloride in water at a temperature of 50 ⁰C is employed as cleaning agent.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS and magnesium chloride in water at a temperature of 35-50 ⁰C.

In an embodiment of the method according to the invention a solution comprising 0.05-5, such as 0.1 % to 1 % SDS plus 0.5 M to 5 M, such as 1 M to 3 M magnesium chloride in water at a temperature of 35 ⁰C - 50 ⁰C is employed as cleaning agent.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising monopropylene glycol in water at a temperature of 50 ⁰C.

In an embodiment of the method according to the invention a solution comprising 10 % to 70 %, such as 20 % to 60 % of monopropylene glycol (MPG) in water at a temperature of 50 ⁰C is employed as cleaning agent.

An embodiment of the invention is a method for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS and monopropylene glycol in water at a temperature of 35-50 °.

In an embodiment of the method according to the invention a solution comprising 0.05-5 %, such as 0.1 % to 1 % SDS plus 10 % to 70 %, such as 20 % to 60 % monopropylene glycol in water at a temperature of 35 ⁰C - 50 ⁰C is employed as cleaning agent.

In an embodiment of the method according to the invention said cleaning solution is of a pH in the range pH 2 - pH 8

EXAMPLE 1

Treatment of protein A adsorbent with selected cleaning agent at various temperatures. Cleaning agent is demineralised water at pH 6.4. A Protein A adsorbent, designed for expanded bed adsorption (UpFront Chromatography A/S, Denmark, Cat. no. XXXX) consisting of cross-linked agarose beads with tungsten carbide particles incorporated as densifying material (final bead density = 3.1 g/ml adsorbent) and protein A (Repligen Corp. CA, USA) immobilised as the adsorption ligand, was treated with demineralised water (the cleaning agent) at various temperatures and various time intervals and then tested for activity of the protein A ligand as follows:

Treatment with cleaning agent:

The protein A adsorbent was initially transferred to a suction filter and washed thoroughly with the cleaning agent and then drained to a wet cake of adsorbent beads. Aliquots of 10 gram wet but drained protein A adsorbent was then transferred into stoppered glass bottles containing 20 ml of the cleaning agent. Different sets of protein A adsorbent submerged in the cleaning agent was then incubated at 50 ⁰C, 60 ⁰C, 70 ⁰C and 80 ⁰C respectively. At various time intervals a single glass bottle with protein A adsorbent was then removed from each of the elevated temperature incubations followed by washing of the adsorbent at room temperature on the suction filter. The washing buffer was in all instances 0.1 M potassium phosphate + 0.1 M sodium chloride pH 7,5 (minimum 200 ml buffer was used for washing).

Determination of static IgG binding capacity:

Following washing, 1.0 ml of adsorbent was transferred into a 10 ml test tube and 8 ml of a solution of highly purified human IgG (Octapharma AG, Schwitzerland, Octagam, cat.no. : 096178, diluted to 7 mg IgG/ml in 0.1 M potassium phosphate + 0.1 M sodium chloride pH 7,5) was added (in total 56 mg IgG added per ml adsorbent). The adsorbent was carefully suspended in the IgG solution and slowly rotated for a period of one hour. The same procedure was followed using a control adsorbent of the same type as the protein A adsorbent, however without the immobilised protein A adsorption ligand (defined as the "non-binding control adsorbent") and the same procedure was also followed using the same protein A adsorbent, which however, had not been treated with the cleaning agent (defined as the "100 % activity control adsorbent"). Following incubation of the adsorbent with purified IgG the adsorbent was allowed to settle and a sample of the supernatant was withdrawn from the test tube for determination of the IgG concentration remaining unbound by measurement of the spectrophotometric absorbance at 280 nm (OD- 280). The absorbance of the supernatant at 280 nm from the different supernatant samples was then used to calculate the amount of IgG bound to the corresponding adsorbent using the absorbance for the supernatant from the non-binding control adsorbent as the reference:

Mg IgG bound/ml adsorbent =

56 x (1 - (OD-280 samp/e/OD-280 non-binding control)).

The binding capacity measured in mg IgG bound per ml adsorbent found for the untreated "100 % activity control adsorbent" was 40 mg IgG/ml protein A adsorbent. Multiple determinations showed a variance of approx. 5 %. The binding capacity of the control was defined to correspond to 100 % activity of the adsorption ligand (protein A). The binding capacity found for all further

(treated) samples was calculated and expressed relative to this 100 % activity control. Thus a binding capacity of 20 mg IgG/ml adsorbent corresponds to a relative binding capacity of 20/40 x 100 % = 50 %; which in turn is defined as a loss of adsorption ligand activity of 50 %.

Figure 1 illustrates the results obtained when treating the protein A adsorbent with demineralised water at 50 ⁰C, 60 ⁰C, 70 ⁰C and 80 ⁰C respectively.

The protein A adsorbent ligand activity is reduced with less than 20 % after 24 hours of treatment at both 50 ⁰C and 60 ⁰C. EXAMPLE 2

The procedure of example 1 was repeated except that the cleaning agent was 0.1 % or 1 % sodium dodecyl sulphate (SDS) in demineralised water at pH 7.2 at 50 ⁰C, 60 ⁰C, and 80 ⁰C respectively.

EXAMPLE 3

The procedure of example 1 was repeated except that the cleaning agent was 10 mM dithiothreitol (DTT) in demineralised water pH 6.5 at 50 ⁰C and 60 ⁰C, respectively.

EXAMPLE 4

The procedure of example 1 was repeated except that the cleaning agent was 1 % sodium lauroyl sarcosinate in demineralised water pH 6.4 at 50 ⁰C.

EXAMPLE 5

The procedure of example 1 was repeated except that the cleaning agent was 20 % 1,2 propanediol (MPG) in demineralised water pH 7.4 at 50 ⁰C and 80 ⁰C, respectively.

EXAMPLE 6

The procedure of example 1 was repeated except that the cleaning agent was 60 % 1,2 propanediol (MPG) in demineralised water pH 7.5 at 70 ⁰C. EXAMPLE 7

The procedure of example 1 was repeated except that the cleaning agent was 1 M sodium chloride in demineralised water pH 7.5 at 50 ⁰C and 60 ⁰C, respectively.

EXAMPLE 8

The procedure of example 1 was repeated except that the cleaning agent was 1 M or 3 M magnesium chloride in demineralised water pH 6.8 at room temperature and 50 ⁰C, respectively.

EXAMPLE 9

The procedure of example 1 was repeated except that the cleaning agent was 0.1 M sodium acetate pH 4.0 or pH 5.0 in demineralised water at 50 ⁰C and 60 ⁰C, respectively.

Claims

1. A method of removal of unbound, entrained and/or non-specifically adsorbed material from an affinity chromatography column comprising the step of contacting said column with a cleaning solution at an elevated temperature with substantial retainment of activity of covalently immobilised adsorption ligands.

2. The method according to claim 1, wherein the elevated temperature is a temperature of at least 35 ⁰C.

3. The method according to claim 2, wherein the elevated temperature is a temperature of at least 40 ⁰C.

4. The method according to claim 3, wherein the elevated temperature is a temperature of at least 50 ⁰C.

5. The method according to claim 4, wherein the elevated temperature is a temperature of at least 60 ⁰C.

6. The method according to claim 5, wherein the elevated temperature is a temperature of at least 70 ⁰C.

7. The method according to any of the preceding claims, wherein the affinity chromatography column is an expanded bed column.

8. The method according to any of the preceding claims, wherein the adsorption ligand is a protein or a proteinaceous material.

9. The method according to claim 8, wherein said protein is Protein A.

10. The method according to any of the preceding claims, wherein the cleaning solution is water.

11. The method according to claim 10, wherein the cleaning solution comprises one or more cleaning agents.

12. The method according to claim 11, wherein said cleaning agents are selected from the group consisting of surface active agents such as cationic, anionic, zwitterionic and non-ionic surfactants, organic solvents such as alcohols, inorganic or organic salts, chaotropic reagents such as chaotropic salts, urea, and guanidinium. hydrochloride, reducing agents, oxidising reagents or any combination of two or more of the above.

13. The method according to any of the preceding claims, wherein the cleaning solution comprises one or more stabilising agents.

14. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with water as a cleaning solution at a temperature of 50 ⁰C.

15. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS in water at a temperature of 50 ⁰C.

16. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising sodium lauroyl sarcosinate in water at a temperature of 50 ⁰C.

17. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising magnesium chloride in water at a temperature of 50 ⁰C.

18. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS and magnesium chloride in water at a temperature of 35-50 ⁰C.

19. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising monopropylene glycol in water at a temperature of 50 ⁰C.

20. The method according to any of the preceding claims for removal of unbound, entrained and/or non-specifically adsorbed material from an expanded bed column comprising the step of contacting said column with a cleaning solution comprising SDS and monopropylene glycol in water at a temperature of 35-50 ⁰C.

21. The method according to any of the preceding claims, wherein said cleaning solution is of a pH in the pH 2-pH 8.