GB2405871A

GB2405871A - Method for controlling protein folding

Info

Publication number: GB2405871A
Application number: GB0321449A
Authority: GB
Inventors: Daniel Brian Jones
Original assignee: Novexin Ltd
Current assignee: Novexin Ltd
Priority date: 2003-09-12
Filing date: 2003-09-12
Publication date: 2005-03-16
Also published as: GB0321449D0; GB0409088D0; GB2405872A

Abstract

A method of removing a folding controlling compound from a protein-containing solution comprises degradation of the compound. Methods of folding or refolding proteins, and methods of controlling protein folding or aggregation, are provided in which the protein is brought into contact with a folding control substance and the folding control substance is degraded. Preferably the folding control substance is a cyclodextrin, and the cyclodextrin is degraded by enzymic digestion by an amylase and/or a glucosyltransferase. The protein may first be denatured by contact with a detergent, chaotrope or reducing agent, prior to refolding in the presence of a cyclodextrin and subsequent degradation of the cyclodextrin. Kits comprising a folding control compound, and solid supports comprising a substance capable of degrading such a compound, are also provided.

Description

M&C Folio: GBP290052 Methods for controlling protein aggregation and

refolding

Technical Field

The present invention relates to methods, consumables, reagents and kits for controlling protein interactions, particularly intra-protein and inter-protein interactions, in particular for controlling protein aggregation, folding and stability.

According to the present invention, denatured protein is contacted with a folding control compound or compounds that maintain the protein essentially in a non- native, non aggregated state; protein folding is carried out in a controlled manner by degrading the folding control compound(s) and thereby affecting the protein structure. Protein folding is carried out at relatively high protein concentration and preferably in the absence of denaturant(s). The present invention also relates to methods for removing folding control compounds from protein solutions.

Background to the invention

The biological function of a protein is dependent on its three dimensional structure. Proteins are formed as linear chains of amino acids (polypeptides).

In viva, in the appropriate conditions within the cell, the linear polypeptide chain is folded, a process which may be assisted by proteins called chaperones. A mature folded protein has an active three dimensional conformation, known as the native structure. The structure depends on weak forces such as hydrogen bonding, electrostatic and hydrophobic interactions. These forces are affected by the protein environment, so changes in the environment may cause structural disruption resulting in denaturation of the protein and loss of function.

The production of proteins by genetic engineering often results in the accumulation of non-active protein aggregates as inclusion bodies. Often, after isolation and purification from the host cells, proteins or inclusion bodies have to be completely unfolded (denatured) and subsequently refolded (renatured) so that the proteins regain their native structure and bioactivity.

Traditional protein folding methods involve denaturant dilution or columnbased approaches. Denatured protein is commonly refolded by diluting the denaturant away. This induces a hydrophobic collapse of the protein molecule and in doing so the protein shields its hydrophobic patches in the core of the molecule.

Unfortunately, on hydrophobic collapse, proteins do not always form the native bioactive conformation; two competing reactions occur: refolding and aggregation. It is suggested that the driver for protein aggregation is hydrophobic amino acid residues exposed at the surface. Aggregation is undesirable and reduces the yield of functional protein. Refolding is known as a first order reaction (Rate = K * [prot]), but the aggregation reaction is favoured over refolding as it is a higher order reaction (n) (Rate = K' * [prot]n). The proportion of protein refolding/aggregation is strongly dependent on the protein concentration. At process scale this concentration dependency results in increased aggregation, and thus reduced refolding yield, due to imperfect mixing patterns. Large protein molecules diffuse more slowly than smaller denaturant molecules, thus creating micro-environments containing high localised protein concentrations and low denaturant concentration, that is, an environment that favours protein aggregation over protein refolding.

For proteins with disulphide bonds the native protein often needs to mature, thus monomeric protein molecules (not aggregated) with non-native disulphide bonds are created initially; then, using a protein specific redox couple, the disulphide bonds shuffle and the protein matures to a functional protein molecule with native disulphide bonds.

Refolding processes usually involve dispersing the denatured protein molecules in a buffer in the presence of "refolding aids" to enhance renaturation. Folding aids usually increase the solubility of the folding intermediates and/or change the relative reaction rates of the folding and aggregation reactions.

Polyethylene glycol and various sugars and detergents have been employed as refolding aids.

Cyclodextrins (CDs) have been reported to be useful in stabilization, solubilisation and affinity purification of certain enzymes, but both the nature of the interactions between CDs and proteins, and their effect on bioactivity remain unclear. Cyclodextrins have been used as artificial chaperones to aid protein refolding in both detergent-free (Sharma et a/, below) and detergent-containing refolding environments (Gellman & Rozema, US 5,563,057). Cyclodextrins are cyclic oligosaccharides composed of multiple glucose residues. They are classified according to the number of sugar residues within the ring structure, a- cyclodextrin has 6 glucose residues, ,B-cyclodextrin has 7 glucose residues and y-cyclodextrin has 8 glucose residues. Cyclodextrin can be modified by derivatisation to produce derivatives.

The inner cavity of Cyclodextrins is hydrophobic whereas the outer surface is hydrophilic. The hydrophobic interior is capable of encapsulating poorly soluble drugs. The hydrophilic exterior assists in solubilisation, so Cyclodextrins are useful adjuncts in pharmaceutical formulation.

EP 0 094 157 & US 4 659 696 (Hirai et a/) describe the use of cyclodextrin derivatives in pharmaceutical compositions consisting essentially of a physical mixture of a hydrophilic, physiologically active (folded, native) peptide and a cyclodextrin derivative, the composition being a uniform mixture in dosage form.

EP 0 437 678 B1, US 5 730 969 and US 5 997 856 (Hora) describe methods for the solubilisation and/or stabilization of polypeptides, especially proteins, using specified cyclodextrin derivatives: hydroxypropyl, hydroxyethyl, glucosyl, maltosyl, and maltotriosyl derivatives of,8- and y-cyclodextrin; the hydroxypropyl--cyclodextrin derivative being preferred. Also disclosed are aqueous and Iyophilised compositions comprising a polypeptide, optionally a protein, and the above specified cyclodextrin derivatives.

EP 0 871 651 & US 5 728 804 (Sharma et al) are concerned with a method for renaturing an unfolded or aggregated protein in a detergent-free aqueous medium with an amount of a cyclodextrin effective to renature said unfolded or aggregated protein. In this instance, the protein is present at a low concentration and spontaneously refolds in a refolding buffer containing cyclodextrin. After refolding the cyclodextrin is removed by dialysis.

There is a need for methods for the efficient preparation of correctly folded, non- aggregated, active protein, particularly proteins produced using recombinant techniques. Control of folding, aggregation and stability of proteins during processing and on storage is a recognised problem in many industries, in particular the pharmaceutical and biotechnology industries. Difficulties encountered with proteins may make manufacture of proteins difficult, result in low yields and render processes uneconomic. Methods, consumables, reagents and kits that permit control of protein refolding, modulate protein aggregation and promote protein stability are commercially important.

Problems addressed by the present invention include reducing protein aggregation and achieving control of protein refolding, in particular of maintaining protein in a stable non-native state, then restoring the original protein properties in a controlled manner to reduce aggregation and thereby increase yield of soluble intermediates or native protein.

Disclosure of invention

The present invention provides a method for removing a folding control compound from an aqueous solution comprising protein, said method comprising degradation of the folding control compound.

The protein in aqueous solution may be denatured, in a fully denatured form or a partially renatured form (i.e. partially folded refolding intermediate); alternatively the protein may be in folded native conformation; the protein solution may contain one or more of these forms. Some protein may also be present in the solution in the form of contaminating aggregates and/or inclusion bodies.

The term "protein" as used herein encompasses proteins, peptides, polypeptides and oligopeptides. Proteins may be synthetic or naturally occurring, and may be obtained by chemical synthesis, or by recombinant or non-recombinant methods.

A folding control compound is a compound which, at an appropriate concentration and in appropriate conditions, can maintain a protein in solution in a non-native or partially-folded soluble state. A folding control compound can be any compound that is capable of shielding hydrophobic amino acid side chains or modifying the net protein charge or hydrogen bonding characteristics and thus altering the protein structure. The concentration of folding control compound used in methods of the invention to maintain protein in a denatured conformation, i.e. in a nonnative or partially folded soluble state, is preferably from 100 to 10, 000 times the molar concentration of protein present, most preferably from 100 to 5,000 times the molar concentration of protein present, yet more preferably from 100 to 2,000 times the molar concentration of protein present, further preferably from 500 to 1,500 times the molar concentration of protein present, most preferably at about 1,000 times the molar concentration of protein present. The folding control compound is preferably a polymer. The term "polymer" encompasses polymeric and oligomeric molecules. A folding control compound is preferably a sugar polymer (oligosaccharide) or a derivative thereof, more preferably a helical or cyclic sugar polymer, yet more preferably a helical sugar polymer such as a fructosan, e.g. an inulin or a derivative thereof; or a cyclic sugar polymer such as glucosan e.g. a cyclodextrin or derivative thereof, most preferably an a-cyclodextrin, a p- cyclodextrin, a y-cyclodextrin, or a derivative thereof. The terms "cyclodextrin" and "cyclodextrin(s)" as used herein include cyclodextrins such as acyclodextrin, a p-cyclodextrin, a y-cyclodextrin and derivatives thereof. One, or more (i.e. a mixture of), folding control compound(s) may be used in methods of the invention.

Degradation of the folding control compound(s) is preferably by chemical degradation and/or by enzymic digestion. Alternatively degradation can be achieved using electromagnetic radiation, shear stress, or heat. When the folding control compound is a sugar polymer or a derivative thereof, degradation is preferably by enzymic digestion, e.g. using carbohydrate degrading enzymes such as amylase, glucosyl transferase, cellulases, or debranching enzymes.

For cyclodextrin(s), degradation is preferably by glucosyltransferase or amylase digestion.

The present invention provides a method for removing a polymer from an aqueous solution comprising protein, said method comprising degradation of the polymer.

The present invention provides a method for removing a sugar polymer from an aqueous solution comprising protein, said method comprising degradation of the sugar polymer.

The present invention provides a method for removing a sugar polymer from an aqueous solution comprising protein, said method comprising enzymic degradation of the sugar polymer.

The invention further provides a method for removing cyclodextrin(s) from an aqueous solution of protein by enzymic degradation of the cyclodextrin(s), preferably by glucosyltransferase or amylase degradation of the cyclodextrin(s).

The present invention provides a method for degrading polymer(s) capable of modifying protein structure comprising: (a) contacting a protein with one or more of said polymer(s), and, (b) degradation of the one or more polymer(s) to reduce or eliminate the structure-modifying properties of the polymer(s).

The present invention provides a method for controlling degradation of polymer(s) capable of modifying protein structure comprising: (a) contacting a protein with one or more polymer(s), and, (b) controlled degradation of the one or more polymer(s) to reduce or eliminate the structure-modifying properties of the polymer(s).

The present invention provides a method for modifying protein structure comprising: (a) in solution, contacting a protein with one or more polymer(s) capable of modifying protein structure, and, (b) degradation of the one or more polymer(s) to reduce or eliminate the structure-modifying properties of the polymer, and thereby modify protein structure.

The present invention provides a method for controlling protein structure comprising: (a) in solution, contacting a protein with one or more polymer(s) capable of modifying protein structure, and, (b) controlled degradation of the one or more polymer(s) to reduce or eliminate the structure-modifying properties of the polymer, and thereby modify protein structure.

Methods according to the present invention also permit folding/refolding of denatured proteins in solution so that properly folded, native conformation protein in solution can be efficiently recovered. Methods of the invention can be applied to virtually any protein, especially after solubilisation and/or denaturation of insoluble protein aggregates, inclusion bodies, or abnormal soluble aggregates.

The invention provides a method for folding protein comprising: (a) contacting denatured protein with one or more folding control compounds(s), and (b) degradation of the folding control compound(s).

The invention provides a method for controlling protein folding comprising: (a) contacting denatured protein with one or more folding control compound(s), and (b) controlled degradation of the folding control compound(s).

The present invention provides a method for folding protein comprising: (a) providing an aqueous solution of denatured protein (unfolded protein and/or partially-folded protein intermediate(s)), (b) contacting denatured protein with one or more folding control compound(s), in an amount, i.e. present at a concentration sufficient to maintain protein in denatured conformation, and (c) degradation of the folding control compound(s), to allow the protein to fold.

The present invention provides a method for folding protein comprising: (a) using a chaotrope, detergent and/or reducing agent to denature protein and/or dissolve aggregated protein and/or inclusion bodies providing an aqueous solution of denatured protein (unfolded protein and/or partially-folded protein intermediate(s)), (b) contacting denatured protein with one or more folding control compound(s) in an amount i.e. present at a concentration sufficient to maintain protein in denatured conformation, (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degradation of the folding control compound(s), to allow the protein to fold.

Suitable chaotropes for use in methods of the invention denature protein and/or dissolve aggregated protein and/or inclusion bodies include guanidine hydrochloride (e.g. 6M), and urea (e.g. 8M).

Suitable reducing agents for use in methods of the invention to denature protein and/or dissolve aggregated protein and/or inclusion bodies include dithiothreithiol, dithioerythritol, beta-mercaptoethanol or Tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HCI), preferably at a concentration in the range of from 1 to 50 mM.

Suitable detergents for use in methods of the invention denature protein and/or dissolve aggregated protein and/or inclusion bodies include SDS, Triton X 100, Sorbitans, e.g. at a concentration of from 0.01 - 2.5% v/v.

The concentration of chaotrope, detergent and/or reducing agent can be reduced by dilution or buffer exchange through dialysis, diafiltration, filtration, chromatographic methods such as gel permeation, size exclusion, chromatography, ion exchange chromatography or affinity chromatography.

Ideally the concentration is reduced to a level that would support the bioactive conformation, in the absence of the folding control compound(s) or any other additive, e.g. typically below 1 M urea or below 0.5 M guanidine hydrochloride.

Protein folding encompasses both folding and refolding. The protein may refold to form a partially folded protein folding intermediate and/or a folded functional protein.

Protein folding conditions (buffer, ionic strength, pH, temperature, redox potential) are highly protein specific. Methods according to the present invention enhance refolding yield in a range of conditions previously known or and may extend the range of conditions that support protein refolding. The reaction conditions, protein concentration and the concentration of the folding control compound are such that initially the protein is essentially maintained in a non-aggregated non-native or partially folded intermediate state, a small amount, preferably less than 5% of the protein may be present in the form of contaminating aggregates and/or inclusion bodies. Removal of the folding control compound by degradation permits refolding of the protein, in a controlled manner, with reduced aggregation.

The concentration of the one or more folding control compound(s) is preferably from 1 to 200 mg/ml, more preferably from 1 to 100 mg/ml, yet more preferably from 10 to 50 mg/ml, further preferably from 10 to 30 mg/ml, most preferably about 20mg/ml.

In prior art methods, refolding is carried out at low protein concentrations, (e.g. for Iysozyme usually less than 0.10 mg/ml). Sharma et a/ describe methods for renaturation of unfolded or aggregated CAB protein that involve contacting the protein in detergent free medium with an amount of cyclodextrin effective to renature the unfolded or aggregated protein. In these methods the protein concentrations are low, selected to minimise aggregation, preferably at around 0.05mg/ml. Refolding occurs in the presence of cyclodextrin and it is suggested that during refolding the relatively polar cyclodextrin molecules that are weakly bound to hydrophobic sites in the folding intermediates are gradually displaced as the interior of the protein becomes increasingly non-polar during refolding.

The cyclodextrins are not removed from the solution to achieve refolding, indeed Sharma states that "Since the cyclodextrins can inhibit aggregation without interfering with protein refolding, they are highly effective protein folding agents". Sharma also advises that that cyclodextrins can be easily separated, if desired, from the refolded protein by dialysis or gel filtration. Thus in methods according to Sharma, when cyclodextrin is removed, it is removed after refolding of the protein.

However, methods, reagents, consumables and kits of the invention, enable protein folding over a range of protein concentrations, at higher protein concentrations than traditional methods and with reduced aggregation. Protein concentrations at which methods of the invention can be performed are generally in the range of from 0.001 to 0.5 mg/ml, preferably in the range of 0.001 to 0.25 mg/ml, more preferably in the range of from 0.001 to 0.15 mg/ml.

The protein concentration chosen will depend on factors such as the size of the protein. Although several smaller proteins have been successfully refolded at higher protein concentrations, generally a lower protein concentration is needed for folding large proteins like antibodies. The advantage of using high protein concentrations is reduced processing volume, resulting in easier purification, reduced reagent usage and reduced capital expenditure (smaller equipment size).

In methods of the present invention, one or more folding control compound(s) (e.g. polymer(s), preferably sugar polymer(s) such as a cyclic or helical sugar polymer(s), more preferably a helical sugar polymer such as fructosan, e.g. an inulin or a derivative thereof; or a glucosan, in particular a cyclodextrin, cyclodextrin derivative or mixture thereof is present in solution initially at a concentration sufficient to prevent complete renaturation and thus maintain protein essentially in a denatured, that is non-native, unfolded or partly folded intermediate conformation. Suitable concentrations of folding control compound or mixture thereof are, for example, in the range of from 1 to 200 mg/ml, preferably from 1 to 100 mg/ml more preferably from 10 to 50 mg/ml, yet more preferably from 10 to 30 mg/ml, most preferably about 20 mg/ml. Suitable concentrations of the one or more polymer(s), sugar polymer; helical or cyclic sugar polymer, fructosan e.g. inulin, or glucosan e.g a cyclodextrin, derivative or mixture thereof are, for example, in the range of from 1 to 200 mg/ml, preferably from 1 to 100 mg/ml more preferably from 10 to 50 mg/ml, yet more preferably from 10 to 30 mg/ml, most preferably about 20 mg/ml. The folding control compound is removed by degradation, preferably controlled degradation. As the folding control compound is degraded, hydrophobic patches on the protein are gradually exposed and refolding can take place. For example, sugar polymer(s), e.g. cyclodextrin(s), derivatives and mixtures thereof, can be degraded in a controlled manner, by chemical or enzymic digestion, permitting controlled folding of the protein. Cyclodextrins can be degraded by amylase or glucosyltransferase digestion.

The invention provides a method for folding protein comprising: (a) contacting denatured protein with one or more cyclodextrin(s), and (b) degradation of the cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion.

The invention provides a method for controlling protein folding comprising: (a) contacting denatured protein with one or more cyclodextrin(s), and (b) controlled degradation of the cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion.

The present invention provides a method for folding protein comprising: (a) providing an aqueous solution of denatured protein (unfolded protein and/or partially-folded protein intermediate(s)), (b) contacting denatured protein with one or more cyclodextrin(s), e.g. by buffer exchange to a solution containing cyclodextrin(s), present in solution at a concentration sufficient to maintain protein in denatured conformation, and (c) degradation of cyclodextrin, preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion to allow the protein to refold.

The present invention provides a method for folding protein comprising: (a) using a chaotrope, detergent and/or reducing agent to denature protein and/or dissolve aggregated protein and/or inclusion bodies to provide an aqueous solution of denatured protein (unfolded protein and/or partially-folded protein intermediate(s)), (b) contacting the denatured protein with one or more cyclodextrin(s) present in solution at a concentration sufficient to maintain protein in denatured conformation, e.g. by buffer exchange to a solution containing cyclodextrin(s), (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degradation of cyclodextrin, preferably by enzyme digestion, to allow the protein to refold.

Denatured protein may be contacted with one or more cyclodextrin(s) by buffer exchange to a solution containing cyclodextrin(s). A concentration of cyclodextrin or cyclodextrins sufficient to maintain protein essentially in denatured conformation is preferably in the range of from 1 to 200 mg/ml, more preferably in the range from 1 to 100 mg/ml yet more preferably in the range of from 10 to 50 mg/ml, further preferably in the range of from 10 to 30 mg/ml, most preferably about 20 mg/ml.

The present invention provides a method for refolding protein comprising: (a) providing a solution of denatured protein using a chaotrope, detergent and/or reducing agent to denature protein and/or dissolve aggregated protein and inclusion bodies, (b) providing to the protein solution of (a) one or more cyclodextrin(s) at a concentration sufficient to maintain the protein essentially in non- native conformation, preferably by buffer exchange to a solution containing one or more cyclodextrin(s), (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degrading the one or more cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion, to allow the protein to refold.

The present invention further provides a method for controlling protein aggregation during folding or refolding comprising: (a) providing a solution of denatured protein, optionally using a chaotrope, detergent, and/or reducing agent to denature protein, (b) providing to the protein solution of (a) one or more cyclodextrin(s), e.g. by buffer exchange to a solution containing one or more cyclodextrin(s), at a concentration sufficient to maintain the protein in non-aggregated form, and (c) reducing the concentration of a chaotrope, detergent, and/or reducing agent, if present, and (c) degrading said one or more cyclodextrin(s) in a controlled manner, by enzyme digestion, preferably by glucosyltransferase or amylase digestion, to induce protein refolding.

A concentration of cyclodextrin sufficient to maintain protein essentially in a non-aggregated form is preferably in the range of from 1 to 200 mg/ml, more preferably in the range of from 1 to 100 mg/ml, yet more preferably in the range of from 10 to 50 mg/ml, further preferably in the range of from 10 to 30 mg/ml, most preferably about 20 mg/ml. A concentration of cyclodextrin or cyclodextrins sufficient to maintain protein essentially in denatured conformation is preferably in the range of from 1 to 200 mg/ml, more preferably in the range from 1 to 100 mg/ml, yet more preferably in the range of from 10 to 50 mg/ml, further preferably in the range of from 10 to 30 mg/ml, most preferably about 20 mg/ml.

Using methods of the invention, the rate of degradation of the folding control compound can be controlled in order to control protein folding or refolding. The rate of glucosyltransferase or amylase digestion of cyclodextrin can be controlled by selecting enzymes with different activity, adjusting the enzyme concentration, solution pH, temperature, ionic strength/composition, redox potential and/or addition of enzyme cofactors. Suitable reaction conditions can be readily determined by those skilled in the art. The benefits of controlling protein folding are the ability to minimise the local concentration of aggregation prone folding intermediates or non-native protein molecules.

In methods of the invention, a disulphide shuffling agent may be present during degradation of the folding control compound. Suitable disulphide shuffling agents for use in methods of the invention include reduced and oxidised glutathione, cysteine, cysteine and disulphide isomerases preferably at a concentration in the range of from 10,uM to 10mM. Disulphide isomerases are preferably used at a concentration of from 1 O, uM to 10 mM.

The invention further provides novel kits and consumables suitable for use in methods of the invention. A kit according to the invention comprises, in a container, one or more folding control compound(s), together with instructions for use of the kit in a method of the invention. A folding control compound is preferably a polymer, more preferably a sugar polymer, more preferably a cyclic or helical sugar polymer, yet more preferably a fructosan such as an inulin or a derivative thereof; or a glucosan such as a cyclodextrin or a derivative thereof.

A kit according to the invention may comprise one or more folding control compound(s) in a first container and a substance or substances capable of degrading the folding control compound(s) in a second container, optionally together with instructions for use of the kit in a method of the invention. A preferred kit according to the invention comprises a cyclodextrin or cyclodextrin(s) in a first container and glucosyltransferase and/or amylase in a second container, optionally together with instructions for use of the kit in a method of the invention. The folding control compound(s) and/or the substance(s) capableof degrading the folding control compound(s) may be provided in (aqueous) solution or in dry form. Thus the cyclodextrin(s) and/or the glucosyltransferase and/or amylase may be provided in aqueous solution or in dry form.

The invention further provides a support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane or column to which is attached a substance or substances capable of degrading a folding control compound or compounds.

The invention further provides a support means, such as a pipette tip or centrifuge tube or column, containing a support to which a substance or substances capable of degrading a cyclodextrin or cyclodextrins is attached.

When the folding control compound is an enzyme, attachment, i.e. immobilization, of the enzyme on the support means and/or support is preferably through covalent bonding. e.g via the amine, thiol, carboxylic acid or aldehyde functional groups of the enzyme.

The invention further provides a support means, such as a pipette tip or centrifuge tube or column, containing a support to which is attached an enzyme or enzymes capable of degrading cyclodextrin.

The invention further provides a support means, such as a pipette tip or centrifuge tube or column, containing immobilized glucosyl transferase or amylase.

The invention further provides a kit comprising one or more folding control compound(s) in a container and a support means such as a pipette tip, centrifuge tube, or column containing a support, such as a resin, gel, beads, or membrane, to which a substance or substances capable of degrading the one or more folding control compound(s) is attached.

The invention further provides a kit comprising one or more folding control compound(s) in a container, and a support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane, or column, to which is attached a substance or substances capable of degrading the one or more folding control compound(s).

The invention further provides a kit comprising one or more cyclodextrin(s) in a container, and a support means, such as a pipette tip, centrifuge tube, or column, containing a support, such as a resin, gel, beads or membrane, to which glycosyltransferase and/or amylase is attached.

The invention further provides a kit comprising one or more cyclodextrin(s) in a container, and a support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane, or column, to which glucosyltransferase and/or amylase is attached.

In methods, consumables and kits of the invention, degradation of the folding control compound can be achieved by contacting the folding control compound- protein solution with, or passing the folding control compound-protein solution through a pipette tip, tube or column containing a substance or substances capable of degrading the folding control compound. The substance(s) may be directly immobilised on the tip tube or column. Alternatively the pipette tip, tube or column may contain the substance(s) immobilised on a resin, gel, bead, membrane or other suitable supporting structure. Degradation of the folding control compound may also be achieved by contacting the folding control compound - protein solution with a resin, gel, bead, membrane, or other suitable supporting structure, to which is attached a substance or substances capable of degrading the folding control compound.

In the methods, consumables and kits of the invention, wherein a cyclodextrin is a, or the, folding control compound, degradation of the cyclodextrin can be by contacting the protein-cyclodextrin solution with, or passing the protein cyclodextrin solution through a glucosyltransferase or amylase-containing pipette tip, tube or column. The glucosyltransferase or amylase enzyme may be directly immobilized on the tip tube or column. Alternatively the pipette tip, tube or column may contain glucosyltransferase and/or amylase immobilised on a resin, gel, bead or membrane or other suitable supporting structure. In a further alternative, degradation of the cyclodextrin can be achieved by contacting the cyclodextrin-protein solution with a resin, gel, bead, membrane or other suitable supporting structure to which is attached glucosyltransferase and/or amylase.

List of Figures Figure 1 shows the aggregation occurring after the breakdown of the cyclodextrin protecting Iysozyme that was initially denatured with urea and then diluted 20x into a cyclodextrin containing phosphate buffer. The y-axis shows the absorbance of red light (500nm). This is a commonly used measure of aggregation. The initial protein concentration was 5 mg/ml in 8M urea. This solution was then diluted 10x into 67mM phosphate buffer at pH 6.5 containing 16.3mM,8-cyclodextrin. The plot shows that,B-cyclodextrin can be broken down by the action of the diastase enzyme to release the aggregation prone Iysozyme refolding intermediates. The higher the diastase concentration the faster the pcyclodextrin breakdown and the greater the rate and overall level of Iysozyme aggregation. The legend indicates the final diastase concentration. The plot shows that the Iysozyme is not completely refolded as the aggregation increases as the,B-cyclodextrin is broken down.

Figure 2 shows the aggregation occurring after Iysozyme denatured in urea was diluted 20x into phosphate buffer. This is the same experiment shown in figure 1 except that the phosphate buffer does not contain p-cyclodextrin. The plot shows that without the protective effect of p-cyclodextrin the overall level of aggregation is increased and no significant change is observed in the aggregation between 50 and 200 minutes as there is no p- cyclodextrin present upon which the diastase enzyme could act. Without p- cyclodextrin, the Iysozyme is not maintained in a denatured form and all of the Iysozyme is available for refolding or aggregation immediately after dilution.

Figure 3 shows the same dilution of denatured Iysozyme into phosphate buffer as figure 1. Each experiment is performed with and without pcyclodextrin in the phosphate buffer. The results have been normalised with the highest A500 value to provide a comparison of the aggregation kinetics. Each curve with,B- cyclodextrin shows a rising A500 value as the Iysozyme is released from the p cyclodextrin to complete the refolding process or to aggregate. The control without p-cyclodextrin present generally show flatter A500 profiles as all the Iysozyme is available for refolding and aggregation immediately after the initial dilution.

Figure 4(a) shows reduction in turbidity between the p-cyclodextrin containing samples and the non--cyclodextrin containing controls at the end of the experiments shown in figure 3. The plot shows an optimum between 0.01 and 1 ug/ml diastase indicating that the rate of,Bcyclodextrin breakdown achieved at this enzyme concentration results in the minimum level of Iysozyme aggregation.

Figure 4(b) shows the enzymatic activity of the Iysozyme samples refolded with B-cyclodextrin. The plot shows an optimum around 1 ug/ml diastase indicating that the total enzyme activity is maximised at the rate of,Bcyclodextrin release achieved by this enzyme concentration. The samples refolded without any,B- cyclodextrin in the refolding buffer did not show any enzymatic activity.

Examples

Experiments were carried out to demonstrate: (i) that 13-cyclodextrin (BCD) can maintain denatured protein in a non- native conformation in an environment that would otherwise, in the absence of BCD, induce both refolding and irreversible aggregation.

(ii) that enzymes can be used to degrade cyclodextrin in proteincyclodextrin complexes in solution.

(iii) that enzymes can be used to induce folding/refolding/aggregation of proteins from BCD/protein complexes.

(iv) that steps (ii) and (iii) can be carried out in a controlled manner whereby the rate of degradation affects both the refolding yield and degree of aggregation.

Example 1: Lysozyme refolding through controlled degradation of cyclodextrin by diastase (amylase) Methodology Aggregation was monitored by measuring the turbidity of a solution, that is, the amount of red light absorbed/scattered by the solution. The more protein aggregates, the higher the turbidity reading. For the experiments discussed below, the turbidity was monitored at 500 nm (A500).

Denatured protein was diluted as described below in 67mM phosphate buffer pH 6.5 containing a high concentration (16.3mM) of p-cyclodextrin (BCD), or in 67mM phosphate buffer pH 6.5 without BCD (control). The turbidity difference after dilution in the different buffers was compared. Maintenance of protein in a non-native non-aggregated conformation by BCD was detected as a reduction in the turbidity relative to controls.

Following this step, a sample of the protein-BCD solution was subjected to enzymatic degradation of the BCD by diastase digestion. An increase in turbidity indicated that both aggregation and refolding were taking place on degradation of BCD, demonstrating that BCD had acted to inhibit protein aggregation and refolding.

Finally an enzyme activity assay was performed to determine the yield of active protein.

Experimental Lysozyme was used a model protein in folding aggregation experiments.

Lysozyme is a monomeric protein of 14.4 kDa. It contains four disulphide bonds. Lysozyme was used because it is a well known, easy to analyse, cheap and readily available protein.

Denatured protein solution A denatured Iysozyme stock of 20 mg/ml was made by dissolving 200 mg of native Iysozyme in 10 ml denaturant buffer containing 8 M urea, 32 mM OTT, 0.1 M Tris, 1 mM EDTA at pH 8.1. This solution was left overnight at room temperature to allow the protein to denature fully. Next, the protein was diluted to a stock of 5 mg/ml denatured protein with denaturant buffer.

BCD solution A BCD solution was made containing 16.3 mM BCD, 10 mM reduced glutathione (GSH) and 1 mM oxidised glutathione (GSSG) in 67mM phosphate at pH 6.5.

Control solution For control experiments (no BCD) a 10 mM GSH and 1 mM GSSG in 67 mM phosphate buffer at pH 6.5 was used.

Diastase solution A diastase stock of 20 mg/ml was made by dissolving 200 mg of diastase in 10 ml of 67 mM phosphate buffer at pH 6.5. A log dilution series was prepared using the same buffer down to a final protein concentration of 20 x 10-7 mg/ml.

Experiment 1 Denatured protein (12.6 ul at 5 mg/ml) was diluted 20x in 239.4,ul BCD (16.3 mM) solution at pH 6.25, or control solution (no BCD) at pH 6.25. Following this, 28'ul of diastase solution was added resulting in a total reaction volume of 280,ul with a final Iysozyme concentration of 0.23 mg/ml and when present, a final BCD concentration of 14.7 mM. This was repeated three times, for each diastase concentration, for both the BCD and control case. The final Iysozyme concentration in the reaction mixtures was 0.23 mg/ml. The turbidity (A500nm) of the samples was measured at 25 minute intervals after dilution (Figure 1, Figure 2). All reactions were carried out at room temperature. The turbidity readings were averaged and normalised to the maximum turbidity reading (Ao) observed for the 'no-BCD' control samples for each diastase concentration (Figure 3).

The difference (delta) between the turbidity of the 'BCD' samples and the 'no BCD' controls corresponds to a reduction of protein aggregation, as shown in figure 3 as the diastase concentration is reduced from 2000 ug/ml, the difference between the CD and no CD curves at 200 minutes increases. The delta turbidity value is the difference between the two curves at 200 minutes expressed as a percentage of the no CD control.

Aggregation in the 'BCD' samples increased on digestion of BCD indicating that the denatured protein had been protected from aggregation in a BCDprotein complex. The rate at which BCD is digested determines the degree of aggregation of protein. For fast degradation i.e. achieved with 2000, ug/ml diastases, no major difference in turbidity was observed. For lower degradation rates, i.e. achieved with 2,ug/ml diastase aggregation was significantly lower in the amylase digested BCD samples when compared to the 'no-BCD' control samples, showing that the pool of soluble nativelike protein was increased by BCD protection of denatured protein, followed by amylase digestion of the BCD.

An optimum degradation rate for BCD, was achieved using 0.02,ug/ml diastase, indicating that controlling the rate of degradation of BCD controls protein refolding and reduces the formation of aggregates, thereby maximising the refolding yield.

Lysozyme activity was measured at room temperature by following the decrease in absorbance at 490 nm of a cell suspension (0.60 mg/ml Micrococcus IysodeiRticus, 67 mM phosphate, pH 6.25). 20 pl of refolded Iysozyme was diluted with 260,ul of 0.60 mg/ml Micrococcus IysodeiEticus in 67 mM phosphate buffer at pH 6.25. The absorbance was observed for 1.5 min. A linear decrease in absorbance was observed. The concentration of refolded Iysozyme was determined by comparing the activity of the refolded Iysozyme to S the activity of standard solutions of native Iysozyme of different concentrations.

For the controls without BCD in the refolding buffer, no Iysozyme activity was observed. For the BCD samples, active, refolded Iysozyme was obtained. The rate of BCD degradation using diastase determines the yield of active Iysozyme (Figure 4).

Claims

CLAIMS: 1. A method for removing a folding control compound from an

aqueous solution comprising protein, said method comprising degradation of the folding control compound.
2. A method according to claim 1 wherein the aqueous solution comprises protein in one or more of a fully denatured form, a partially folded form; a native conformation, an aggregated form or an inclusion body.
3. A method for folding protein comprising: (a) contacting denatured protein with one or more folding control compounds(s), and (b) degradation of the folding control compound(s).
4. A method for controlling protein folding comprising: (a) contacting denatured protein with one or more folding control compound(s), and (b) controlled degradation of the folding control compound(s).
5. A method for folding protein comprising: (a) providing an aqueous solution of denatured protein, (b) contacting denatured protein with one or more folding control compound(s) present at a concentration sufficient to maintain protein in denatured conformation, and (c) degradation of the folding control compound(s), to allow the protein to fold.
6. A method for folding protein comprising: (a) using a chaotrope, detergent, and/or reducing agent to denature protein and/or dissolve aggregated proteins and/or inclusion bodies to provide an aqueous solution of denatured protein, (b) contacting denatured protein with one or more folding control compound(s) present at a concentration sufficient to maintain protein in denatured conformation, (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degradation of the folding control compound(s), to allow the protein to fold.
7. A method according to claim 6 wherein the chaotrope is one or more of guanidine hydrochloride and/or urea.
8. A method according to claim 6 wherein the reducing agent is one or more of dithiothreithiol, dithioerythritol, beta-mercaptoethanol or Tris (2carboxyethyl) Phosphine Hydrochloride (TCEP.HCI).
9. A method according to claim 6 wherein the detergent is one or more of SDS, Triton X 100, or a Sorbitan.
10. A method according to any one of claims 6 to 9 wherein the concentration of chaotrope, detergent and/or reducing agent is reduced by dilution or buffer exchange through dialysis, diafiltration, filtration, and/or a chromatographic method, such as gel permeation, size exclusion, chromatography, ion exchange chromatography and/or affinity chromatography.
11. A method according to any preceding claim wherein the folding control compound is a polymer.
12. A method according to any preceding claim wherein the folding control compound is a sugar polymer or a derivative thereof.
13. A method according to any preceding claim wherein the folding control compound is a cyclic sugar polymer.
14. A method according to claim 13 wherein the folding control compound is a glucosan.
15. A method according to claim 14 wherein the folding control compound is a cyclodextrin or derivative thereof.
16. A method according to claim 15 wherein the cyclodextrin is a an acyclodextrin, a,8-cyclodextrin, a y-cyclodextrin, or a derivative thereof.
17. A method according to claim 12 wherein the folding control compound is a helical sugar polymer.
18. A method according to claim 17 wherein the folding control compound is a fructosan.
19. A method according to claim 18 wherein the folding control compound is an inulin or a derivative thereof.
20. A method according to any preceding claim wherein the concentration of folding control compound in (b) is from 100 to 10,000 times the molar concentration of protein.
21. A method according to any preceding claim wherein degradation of the folding control compound(s) is by one or more of the following methods: chemical degradation, enzymic digestion, electromagnetic radiation, shear stress, or heat.
22. A method according to any one of claims 12 to 21 wherein degradation is by enzymic digestion.
23. A method according to claim 22 wherein degradation is by glucosyltransferase or amylase digestion.
24. A method according to any preceding claim wherein the concentration of folding control compound or mixture thereof is in the range of from 1 to 200 mg/ml.
25. A method according to claim 24 wherein the concentration of folding control compound or mixture thereof is from 1 to 100 mg/ml.
26. A method according to claim 25 wherein the concentration of folding control compound or mixture thereof is from 10 to 50 mg/ml.
27. A method according to claim 26 wherein the concentration of folding control compound or mixture thereof is from 10 to 30 mg/ml.
28. A method according to claim 27 wherein the concentration of folding control compound or mixture thereof is about 20 mg/ml.
29. A method for folding protein comprising: (a) contacting denatured protein with one or more cyclodextrin(s), and (b) degradation of the cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion.
30. A method for controlling protein folding comprising: (a) contacting denatured protein with one or more cyclodextrin(s), and (b) controlled degradation of the cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion.
31. A method forfolding protein comprising: (a) providing an aqueous solution of denatured protein, (b) contacting denatured protein with one or more cyclodextrin(s) present in solution at a concentration sufficient to maintain protein in denatured conformation, and (c) degradation of the cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion, to allow the protein to refold.
32. A method for refolding protein comprising: (a) using a chaotrope, detergent, and/or reducing agent to denature protein and/or dissolve aggregated proteins and/or inclusion bodies to provide an aqueous solution of denatured protein, (b) contacting denatured protein with one or more cyclodextrin(s) present at a concentration sufficient to maintain protein in denatured conformation, (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degradation of the cyclodextrins(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion, to allow the protein to refold.
33. A method for refolding protein comprising: (a) providing a solution of denatured protein using a chaotrope, detergent and/or reducing agent to denature protein and/or dissolve aggregated protein and inclusion bodies, (b) providing to the protein solution of (a) one or more cyclodextrin(s) at a concentration sufficient to maintain the protein essentially in nonnative conformation, preferably by buffer exchange to a solution containing one or more cyclodextrin(s), (c) reducing the concentration of the chaotrope, detergent and/or reducing agent, and (d) degrading the one or more cyclodextrin(s), preferably by enzyme digestion, most preferably by glucosyltransferase or amylase digestion, to allow the protein to refold. 30
34. A method according to claim 32 or 33 wherein the chaotrope is one or more of guanidine hydrochloride and/or urea.
35. A method according to claim 32 or 33 wherein the reducing agent is one or more of dithiothreithiol, dithioerythritol, beta-mercaptoethanol or Tris (2 carboxyethyl) phosphine hydrochloride (TCEP.HCI).
36. A method according to claim 32 or 33 wherein the detergent is one or more of SDS, Triton X 100, or a Sorbitan.
37. A method according to any one of claims 33 to 36 wherein the concentration of chaotrope, detergent and/or reducing agent is reduced by dilution, or buffer exchange through dialysis, diafiltration, filtration, and/or a chromatographic method, such as gel permeation, size exclusion, chromatography, ion exchange chromatography and/or affinity chromatography.
38. A method for controlling protein aggregation during folding or refolding comprising: (a) providing a solution of denatured protein, (b) contacting denatured protein with one or more cyclodextrin(s), present in solution at a concentration sufficient to maintain the protein in non aggregated form, and (c) degrading said one or more cyclodextrin(s) in a controlled manner, by enzyme digestion, preferably by glucosyltransferase or amylase digestion, to induce protein refolding.
39. A method according to any preceding claim wherein a disulphide shuffling agent is present during degradation of the folding control compound.
40. A method according to claim 39 wherein the disulphide shuffling agent is selected from the group comprising a reduced or oxidised glutathione, cysteine, cysteine or disulphide isomerases.
41. A kit comprising, in a container, one or more folding control compound(s), together with instructions for use of the kit.
42. A kit comprising one or more folding control compound(s) in a first container and a substance or substances capable of degrading the folding control compound(s) in a second container, optionally together with instructions for use of the kit.
43. A kit comprising a cyclodextrin or cyclodextrin(s) in a first container and glucosyltransferase and/or amylase in a second container, optionally together with instructions for use of the kit.
44. A support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane or column to which is attached a substance or substances capable of degrading a folding control compound or compounds.
45. A support means, such as a pipette tip or centrifuge tube or column, containing a support to which is attached a substance or substances capable of degrading a cyclodextrin or cyclodextrins.
46. A support means, such as a pipette tip or centrifuge tube or column, containing a support to which is attached an enzyme or enzymes capable of degrading cyclodextrin.
47. A support means, such as a pipette tip or centrifuge tube or column, containing immobilized glucosyl transferase or amylase.
48. A kit comprising one or more folding control compound(s) in a container and a support means such as a pipette tip, centrifuge tube, or column containing a support, such as a resin, gel, beads, or membrane, to which is attached a substance or substances capable of degrading the one or more folding control compound(s).
49. A kit comprising one or more folding control compound(s) in a container, and a support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane, or column, to which is attached a substance or substances capable of degrading the one or more folding control compound(s).
50. A kit comprising one or more cyclodextrin(s) in a container, and a support means, such as a pipette tip, centrifuge tube, or column, containing a support, such as a resin, gel, beads or membrane, to which glycosyltransferase and/or amylase is attached.
51. A kit comprising one or more cyclodextrin(s) in a container, and a support, such as a pipette tip, centrifuge tube, resin, gel, beads, membrane, or column, to which glucosyltransferase and/or amylase is attached.