Chromatographic refolding of proteins: molecular action and column control

CHINA PARTICUOLOGY Vol. 3, No. 6, 337-342, 2005

CHROMATOGRAPHIC REFOLDING OF PROTEINS: MOLECULAR ACTION AND COLUMN CONTROL Fangwei Wang1,2, Yongdong Liu2 , Jing Chen2 and Zhiguo Su2,* 1 Bioscience and Biotechnology Department, Dalian University of Technology, Dalian 116024, P. R. China National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100080, P. R. China * Author to whom correspondence should be addressed. E-mail: [email protected]

2

Abstract Protein expression in E. coli often results in the formation of a kind of protein aggregate called inclusion body. Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products. Conventional dilution refolding technique suffers from disadvantages of low recovery and low concentration. Various chromatographic refolding techniques have been developed over the last few years. These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to refold at nano-scale. The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation. Keywords

protein refolding, size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography

1. Introduction The formation of protein aggregates is a big problem in the processing of protein products. These aggregates are produced in fermentation, in separation, in storage, or in application. Aggregates contain proteins with incorrect three dimensional structures, and without the desired biological activity. A way to restore the native biological activity is to dissolve the aggregate and let the protein refold. Such an operation is called protein refolding. It has become a unit operation in biotechnology industry to obtain genetic engineered proteins from microorganisms, notably E. coli (Misawa & Kumagai, 1999). Protein expression in E. coli often results in the formation of a kind of protein aggregate called inclusion body (IB). IB can protect the protein against the attack by the host cell, and can be easily separated from fermentation broth by simple centrifugation or filtration. However, there is no biological activity for the protein in IB. The challenge for bioprocess engineers is how to convert the inactive and insoluble IB protein aggregate into soluble, correctly folded and biologically active protein (De Bernadez Clark, 2001). Figure 1 is a schematic picture of IB protein conversion. The photo at upper left corner in Fig. 1 is an E. coli cell containing IB particles. In general, two steps are involved in converting the inactive protein into active one. First, the IB has to be solubilized in a denaturant solution. This can be done by high concentration urea or guanidine-HCl together with some reducing agent such as dithiotheritol to break up incorrect disulphide bonds. The solubilized protein in urea or guanidine-HCl solution is in denatured form, with unfolded, stretched peptide chain without any biological activity. The second step is to initiate folding of the peptide chain of the protein by taking the denaturant molecules away from the unfolded chain. This is a thermodynamically driven process in which the stretched peptide chain starts to figure itself through molecular action to

form α helix, β- sheet, β-bend or β-turn. This self folding process may result in the formation of correct three dimensional structure of the native protein. The second step is a crucial one that decides the success of refolding because misfolded or aggregated proteins can also occur in large quantities, drastically reducing the recovery of active protein, which can be as low as 5-10%. In some cases, there could be no active protein found.

Inclusion body Inclusion body

Step 1: 1: Step solubilization solubilization

Fig. 1

Stretched peptide Stretched peptide

Active protein Active protein

Step 2:2: Step Removal denaturant Removal of of denaturant

A schematic picture of inclusion body protein conversion.

Various strategies for the second step have been developed. Dilution is the most commonly used method, which is depicted in Fig. 2. The urea or guanidine-HCl solution containing unfolded protein is diluted with buffers to reduce its concentration. The dots surrounding the peptide chain are imitations of denaturant molecules. Because of the dilution, there are much less denaturant molecules surrounding the peptide chain. The unfolded peptide chain, which is a polymer of different amino acids consisting of different side groups, can then fold through intra-molecular interactions such as ionic, hydrophobic, van der Waals among different amino acid side groups located at different places of the polymer chain.

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Soluble Soluble denatured denatured protein protein

Dilution refolding Dilution refolding

1:10–200 1:10-200

Fig. 2

Dilution refolding process with a refolding buffer.

Figure 3 shows the formation of active protein with refolding time under different dilution folds (Li et al., 2004). Apparently the more the dilution folds, the higher the specific activity. However, dilution consumes a lot of buffer and increases the volume of the production stream. For exam3 ple, if the dilution fold is 100, then 1 m solution will become 3 100 m solution for further processing, a big burden for downstream processing. An additional problem is that the product concentration has become very low, which is not suitable for further processing such as chromatography. Therefore extra steps have to be taken to concentrate the solution, which further decreases the recovery.

-1 Specific Specificactivity activity(U/mg) / U.mg

14000 14000 12000 12000 200 *200 100 *100 10 *10

10000 10000 8000 8000 6000 6000 4000 4000 2000 2000 00 00

100 100

200 200

300 300

400 400

Time (min) Time / min Fig. 3

The specific activity of lysozyme with time during dilution re-1 folding. Denatured lysozyme in 8 mol.mL urea, protein -1 . concentration 6 mg mL . The three curves denote 10, 100 and 200 times of dilution.

Unfoldpeptide(U) peptide (U) Unfold

↔I II1↔ II2↔· ·····↔CC C

UU U

11

2

↕

M M

Inter mediate(I) Intermediate (I)

Misfold(M) Misfold(M) Correct(C) Correct (C) Aggregate(A) Aggregate (A)

Fig. 4

AA

Possibilities: Possibilities: M+M ↔ A M+M↔A M+I ↔ A M+I↔A M+C ↔ A M+C↔A I+C ↔ A I +C↔A

Analysis of the refolding pathway.

Although the specific activity of lysozyme in Fig. 3 reached almost 12,000 U.mg-1, it was far less than its original native activity of 42,000 U.mg-1. In other words, there were misfolding and aggregation of the protein that accounted for more than 2/3 of the total protein. Fig. 4 is an analysis of the refolding pathway. Unfolded protein (U) undergoes an intermediate (I) before becoming an active product. The intermediate, however, could form three structures which are correct structure (C), misfolded structure (M), and aggregate (A). In a real process, there could be more intermediates as I1, I2, etc. which might deviate from the correct path to M and A. There are also possibilities of aggregates formation through interaction between M, I and even C. Therefore, the refolding pathway is really complicated. To increase the productivity of the C, it is essential to inhibit misfolding and aggregation (Gu et al., 2004). Recent studies have found that certain low molecular weight additives are possible to inhibit the intermolecular interactions that cause aggregation in dilution refolding (Lilie et al., 1998). Commonly used additives are L-arginine (0.4-1 M), low concentration of denaturants such as urea (1-2 M) and guanidine-HCl (0.5-1.5 M), and detergents (CHAPS, CTAB, and Triton X-100). High hydrostatic pressures (1-2 kbar) in combination with low concentrations of denaturants have been used for simultaneous solubilization and refolding of inclusion body proteins (Foguel et al., 1999). In spite of the progress, the inherent disadvantage of dilution refolding remains. Over the last few years, various chromatographic refolding techniques have been developed in several labs including the authors’ one. Chromatography has been a very successful technique in protein separation and purification. It uses a solid medium, usually porous microspheres, packed in a cylindrical column. The feed solution is introduced from one end of the column, moving forward with various liquid solutions. Different substances in the feed are separated during the process. The substance having the least interaction with the solid medium exits the column first, while the substance having the strongest interaction with the medium exits the last. In this way, different proteins can be separated and purified. Like in protein separation and purification, chromatographic refolding also utilizes the solid media in a similar way, but the purpose and the control strategy are different. In chromatographic refolding, the solid medium acts as a kind of chaperone or assistant to help the protein refolding in a correct way, which minimizes misfolding and aggregation. Fig. 5 is a simplified illustration of chromatographic refolding. The feed solution containing denatured protein and denaturant is loaded into the column packed with porous microspheres. Renaturation buffers are introduced to elute the denatured protein to move through the column. During this process, simultaneous refolding and adsorption take place. The solid phase helps the correct folding of the protein. At the column outlet, the protein exits in correct form.

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Wang, Liu, Chen & Su: Chromatographic Refolding of Proteins: Molecular Action and Column Control

A simplified illustration of chromatographic refolding.

Chromatographic refolding is a very complicated process. There are intra-molecular folding, inter-molecular interaction between the protein and the ligand on the solid phase, and macroscopic process control over the entire column. The size of protein is several nanometers but the microsphere is several tens of micrometers. The length of the column can be more than half a meter. At this moment, it is difficult to describe the mechanism of chromatographic refolding, because the information available is very limited. Here the molecular action and column control strategy in three types of chromatographic refolding processes are discussed.

2. Refolding with Size-Exclusion Chromatography (SEC) SEC is a successful tool for protein purification. In the case of protein refolding, two key factors need consideration (Batas & Chaudhuri, 1999). The first is the loading of the protein to the column in the presence of a denaturant solution. The second is the change in protein size that occurs as it renatures during elution with the refolding buffer. Fig. 6 is an illustration of the fundamental principle for SEC refolding. The pores of the SEC media have selectivity on the size and shape of the proteins. Unfolded protein is a stretched long shape which is difficult to get into the pores. The correctly formed product, being compact in shape and size, could get access into the depth of the pores, thus separating itself from unfolded and partially folded ones.

gradient to provide a gentle and easily controllable environment for protein refolding. In this process a quick change in urea concentration was avoided. The procedure was gentle, providing a gradual change of the environment for the protein to refold gradually. A refolding study with lysozyme as a model protein using the gradient SEC has been reported (Gu et al., 2001). In another study (Gu et al., 2002), a recombinant scFv fusion protein expressed as inclusion bodies in E. coli was refolded on a HiLoad 16/60 Superdex 30 prep-grade column. Fig. 7 is a comparison of different refolding methods. Dilution refolding gave less than 10% recovery. SEC without gradient could increase the recovery to more than 20%. Much better results were found with gradient refolding, especially with the dual-gradient of decreasing denaturant concentration and increasing pH. The activity recovery exceeded 50%. The principle of this method is that before feed loading, the column is equilibrated with the refolding buffer, followed by the introduction of a descending gradient of denaturant (e.g. from 6M Gu-HCl or 8M urea down to a predetermined concentration in the refolding buffer), sometimes combined with an increasing pH-gradient. The gradient is allowed to occupy the upper 60% of the column. The feed in the highest denaturant concentration is then added, followed by a small volume of the same denaturant concentration in order to avoid uncontrolled dilution of the protein in the rear part of the feed zone. During the elution the proteins will be restricted to the void volume only, implying that they will pass through regions with gradually decreasing denaturant concentration, reaching the final refolding buffer concentration just before leaving the column. Specific activity Activity recovery 60

60

40

40

20

20

0

a

b

c

d

Activity recovery / %

Fig. 5

Loading solution Loadingsolution solution Elution Elution solution

Specific activity / %

Denatured protein Denatured protein

0

Refolding methods Fig. 7 Refolding of a single chain antibody scFv57P with different methods. a: Dilution; b: SEC without gradient; c: SEC with denaturant gradient; d: SEC with dual gradients of denaturant and pH. The specific activity is relative to the final purified protein’s specific activity. Fig. 6

Principle of protein refolding with size exclusion chromatogramphy. The stretched peptide could not enter the pore of the microsphere, while the refolded one could get inside due to compact structure.

A new SEC refolding concept was introduced. It was a gradient refolding system based on a decreasing urea

3. Refolding with Ion Exchange Chromatography (IEC) A very efficient strategy to prevent aggregation is to minimize the chance of intermolecular interactions. This could be achieved by adsorbing the denatured protein

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molecules onto a solid support, thus effectively separating the individual protein molecules during refolding. IEC, a widely used process for protein separation, can work for this purpose. However, refolding with IEC should be careful. Figure 8 is an illustration for IEC refolding. The denatured protein is adsorbed onto the surface of IEC medium. As the concentration of denaturant in the liquid phase decreases, the protein starts to fold. The folding could take place on the solid surface or away from the surface depending on the protein and its environment. Partial folded protein could also be adsorbed by the solid surface through ionic interaction. The ideal situation is that the protein finishes its refolding process to form a correct structure, and to exit the column. Because the adsorption of protein could separate individual molecules, their refolding is not interfered by each other, and the chance of aggregation is minimized. Adsorption Adsorption

Folding & adsorption Folding & adsorption

Elution Elution

Change of Change of denaturant denaturantconcentration concentration Fig. 8

Principle of protein refolding with adsorption chromatography. The process is an imitation for ion exchange chromatography and hydrophobic interaction chromatography.

In one report, refolding of bovine-lactalbumin with IEC gave less than 10% recovery, and the best result for hen lysozyme was also as low as 10% (Creighton, 1986). The soluble refolding intermediates bound tightly to the medium and were very difficult to elute from the column. In order to prevent accumulation of non-eluted protein during refolding of matrix adsorbed protein molecules, a new process using a two-buffer system to improve activity yield and mass recovery has been developed (Li et al., 2002). The feed was loaded into the ion exchanger and the protein adsorbed onto the solid media in the presence of 8M urea. A descending urea concentration gradient was introduced in parallel with an increasing ionic strength, allowing the protein to simultaneously structurally rearrange and elute during its migration down through the column. When it entered regions where the salt concentration was low, the protein would be adsorbed onto the ion exchanger again. The salt concentration increased and the urea concentration decreased gradually in the column, leading to desorption of the protein. Finally, at the column outlet, the urea concentration was 1M and the salt concentration was high enough for the protein to be eluted. This type of “on/off” cascade process would allow a single protein molecule to refold gradually without much chance of intermolecular in-

teraction and facilitate the refolding of the protein to a native, biologically active conformation. Another factor to be considered in optimizing a refolding process, especially in the formation of disulfide bonds, is the pH of the refolding buffer (Misawa et al., 1994). The most favorable pH value varies from protein to protein. Usually, aggregation decreases when the pH of the medium is far away from the protein’s isoelectric point (Cleland, 1993). The effect of nearby charged residues on the oxidation potential also makes a difference (Zhang & Snyder, 1989). In order to accelerate the thiol-disulfide exchange, the pH of the renaturation buffer should be at the upper limit that still allows the protein to form its native structure. However, it may be difficult to optimize denaturant concentration and pH simultaneously in a refolding process, especially in a large-scale production. Considering the importance of both denaturant concentration and pH in refolding, a dual-gradient IEC process was introduced to enhance the refolding recovery at high protein concentration (Li & Su, 2002a). After the dissolved human lysozyme expressed as inclusion bodies was loaded into the column, elution was started by gradually decreasing the urea concentration, combined with a gradual increase of pH of the elution buffer. The dual-gradient provides an incremental change of the solution environment for the protein refolding and for the formation of disulfide bonds. Fe-SOD, that is lack of disulfide bonds, showed an increased refolding yield when a dual-gradient IEC refolding process was applied (Li & Su, 2002b). At high pH, far away from the protein’s isoelectric point, aggregate formation was prevented, while at low pH near the isoelectric point the establishment of a biologically active conformation was facilitated.

4. Refolding with Hydrophobic Interaction Chromatography (HIC) High performance hydrophobic interaction chromatography (HIC) was used to refold recombinant human interferon. Refolding and purification could be achieved in one step. The refolding yield was twice as high as that obtained using dilution or dialysis (Guo, 2001). Reverse phase high performance liquid chromatography (RPC) as a refolding tool could refold recombinant human interleukin-2 expressed as inclusion bodies. The total activity recovery and specific activity were increased 9 and 14 folds, respectively (Ling et al., 1997). However, successful polypeptide folding is also dependent on undisturbed hydrophobic interaction forces. This is why HIC or RPC interactions should not be as strong as to prevent proper protein refolding. Some binding strength modifying agents might reduce the hydrophobic interaction and improve the refolding when added to the refolding buffer. The additives may influence both the solubility and the stability of the native, denatured and in-

Wang, Liu, Chen & Su: Chromatographic Refolding of Proteins: Molecular Action and Column Control termediate states, respectively. They may act by changing the ratio of the rates of proper folding and aggregate formation or they might simply act by solubilizing aggregates already formed. A new potent system adopting hydrophobic interaction chromatography (HIC) assisted by glycerol was utilized to refold lysozyme at high initial protein concentration (Li et al., 2004a). Denatured and reduced lysozyme of 50 mg.mL-1 as a model protein was loaded to Poros PE perfusion column, a commercially available HIC support, with a ratio of 1mg protein per mL HIC medium adopting a gradient of urea and salt. Another gradient of urea and glycerol was utilized to deprive bound lysozyme from HIC and then refold it, resulting in activity yield of more than 85% with native special activity. HIC could distinctly enhance the protein recovery simultaneously minimizing aggregate due to its capability of binding unfolded and partially folded proteins when used to refolding proteins, but it was prone to decrease special activity of refolded protein, hinting the formation of incorrect refolded structure. Glycerol was able to facilitate the formation of native structure of lysozyme on HIC due to its hydration ability for refolding proteins accompanied by the adsorption of proteins with urea gradient. The concentration of glycerol, urea gradient volume, ionic strength and the concentration of reducing reagent of mercaptoethenol were found to play an important role on the activity yield. A very mild hydrophobic ligand of polyethylene glycol immobilized onto agarose microspheres is used successfully for refolding of recombinant Staphylococcus aureus Elongation Factor G (Li et al., 2004b).

5. Refolding with Other Chromatographic Processes Immobilized metal-ion affinity chromatography (IMAC) has opened new prospects for efficient purification and refolding of proteins equipped with engineered polyhistidine tags. Polyhistidine tags form high affinity complexes with immobilized divalent metal ions even in the presence of high concentrations of chaotropic agents, thereby allowing isolation and refolding of tagged protein. Thus, one-step on-column affinity refolding and purification processes have become quite popular (Glynou et al., 2003). As in the IEC refolding process, a gradual decrease of denaturant concentration induces protein refolding. Elution is achieved by increasing the imidazole concentration or by using a decreasing pH gradient (Zouhar et al., 1999). Thus the fusion protein His-TNF expressed in E. coli as inclusion bodies was refolded after adsorption to a 2+ Ni -Sepharose 6B column, resulting in a more than 90% refolding yield (Xu et al., 2000). Affinity chromatography (AFC) using various ligands can also be used to refold proteins. For example, the strongly negatively charged Heparin-Sepharose was used for the binding of a denatured protein containing a polyarginine fusion tag. Renaturation could be achieved under conditions allowing the protein to remain bound to the matrix and resulted in high yields of active protein (Stempfer et al., 1996).

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Chaperones GroEL and GroES can bind to nascent or unfolded polypeptides and/or their folding intermediates, preventing improper polypeptide chain interactions that lead to aggregation. It is not surprising that these proteins can also affect the in vitro competition between folding and aggregation (Mayer et al., 2000). Because chaperones and foldases are proteins that need to be removed from the renaturation solution at the end of the refolding process and as they may be costly to produce, their commercial use will require a recovery-reuse scheme (Misawa & Kumagai, 1999). GroEL immobilized to agarose gel has been utilized in a lysozyme refolding study (Dong et al., 2000). Like in protein separation and purification, chromatographic refolding can be carried out with combination of different chromatographic modes as IEC, IMAC, SEC etc. An example is refolding of NS3，a potential target protein for HCV therapy (Li et al., 2003). The expression of full-length NS3 in E. coli results primarily in the formation of inactive aggregates (Kolykhalov et al., 2000). Because it has a histidine tag, partially purification and refolding were initially performed with IMAC after dissolution of the inclusion body in 8M urea. The IMAC process separated the denatured NS3 from other impurities present in the inclusion body. It also refolded the protein to its intermediate state. A further refolding and purification were followed either with IEC or with SEC, or even with another IMAC. The comparative results for the second chromatographic refolding process demonstrated that SEC was the best choice since it did not require a pre-desalting procedure like in the case of IEC after the first IMAC process. Combination of IMAC and SEC chromatographic steps gave 90% of the total activity recovery.

6. Conclusion Conversion of inactive misfolded protein into active product is a very complicated process. Dilution refolding can be replaced by chromatographic refolding to increase the product recovery and concentration. The selection of chromatographic media and process is a key factor to determine the success of the new technique. There are intra-molecular folding at nano-scale, inter-molecular interaction between the protein and the ligand on the solid phase of micro-scale, and macroscopic process control over the entire column of meter-scale. An optimized gradient elution could meet the multi-scale requirement and result in significant improvements.

Acknowledgement The Natural Science Foundation of China (NSFC No.20136020, 20125616) and Chinese Academy of Sciences are gratefully acknowledged for financial supports to this research.

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