WO1994002625A1

WO1994002625A1 - Process for isolating recombinant polypeptides

Info

Publication number: WO1994002625A1
Application number: PCT/US1992/006069
Authority: WO
Inventors: Per Wikstrom
Original assignee: Immunex Corporation
Priority date: 1992-07-21
Filing date: 1992-07-21
Publication date: 1994-02-03
Also published as: AU2383392A

Abstract

There is disclosed a process for isolating a recombinant polypeptide produced as a component of an insoluble fraction in a transformed host cell comprising entrapping the host cells within a porous matrix; disrupting the host cell membrane to enable diffusion of soluble host cell proteins from the host cell; washing the entrapped host cell to remove the soluble host cell proteins while leaving the insoluble fraction within the porous matrix; and contacting the insoluble fraction within the porous matrix with a chaotropic agent to isolate the recombinant polypeptide from the insoluble fraction.

Description

PROCESS FOR ISOLATING RECOMBINANT POLYPEPTIDES

TECHNICAL FTELD OF THE TNVENTTON

This invention relates generally to methods for isolating and purifying recombinant polypeptides. More particularly, this invention relates to a method for isolating recombinant polypeptides from transformed prokaryotic host cells utilizing a porous matrix to entrap insoluble material.

BACKGROUND OF THE INVENTION

Recombinant polypeptides are expressed as heterologous proteins in transformed host cells. The transformed host cells may be prokaryotic or eukaryotic in origin. The recombinant polypeptides are made by the host cell's protein machinery according to the structural gene that is transfected into the host cell genome. Following translation, the host cell may excrete the recombinant polypeptide or accumulate it intracellularly. Recovery of recombinant polypeptides that accumulate within a host cell may be difficult and commercially impractical on a large scale.

When producing recombinant proteins, such as IL-lβ, it is a difficult exercise to separate expressed polypeptide from transformed host cells or their culture supematants (Dwyer, Bio/Technology 2:1957 (1984)). Mammalian polypeptides expressed in prokaryotic or bacterial cells occasionally form precipitates (which are also sometimes referred to as "aggregates," "refractile bodies," or "inclusion bodies"). See, e.g., United States Patent 4,569,790; Langley et al., Eur. J. Biochem. 763:313 (1987); Winkler et al., Bio/Technology 3:990 (1985); and DeLamarter et al., EMBO J. 4:2575 (1985). Such aggregates or inclusion bodies are associated with formation of intermolecular or intramolecular bonds, or hydrogen bonds and disulfide bonds having a deleterious effect upon biological activity.

Current techniques to recover recombinant polypeptides which accumulate intracellularly as inclusion bodies typically include a procedure to break open the cells, followed by one or more centrifugation steps to separate insoluble cellular debris from soluble proteins. Insoluble material forms a cell pellet. The cell pellet is composed of cellular wall material and often insoluble recombinant polypeptide. After removing the supernatant, the recombinant polypeptide must then be extracted in a long and arduous procedure from the cellular debris in the pellet.

Separating a recombinant polypeptide (in a soluble or an insoluble form) from other host cell materials is difficult and usually involves centrifugation. Centrifugation is a difficult process for large-scale purification of recombinant polypeptides because centrifugation is generally conducted batchwise and not continuously. Moreover, centrifugation to separate soluble from insoluble materials in the presence of a high concentration of a chaotropic agent, such as 7 M guanidine HC1, is frequently unsuccessful. Therefore, it is desirable to avoid centrifugation in large-scale purification processes and allow continuous processing of recombinant polypeptides.

For example, European Patent Application 200,986 describes a procedure for isolating human recombinant interleukin-lα from E. coli. An E. coli paste (1 g) was suspended in 5 ml. of 1 mM phenylmethylsulfonyl fluoride in buffer A (30 mM Tris- HC1, pH 8, 5 mM EDTA) and the cells were sonicated six times for a total of 3 minutes. The cell lysate was centrifuged for 30 min. at 30,000 X g to separate the insoluble fraction. The paniculate fraction (which contained the IL-lα activity) was sequentially washed with with 5 ml each of: 1) buffer A, 2) 1% Triton X- 100 in buffer A, and 3) 1.75 M guanidine HC1. After each washing, the paniculate fraction was pelleted by centrifugation at 30,000 X g for 20 min. IL-lα polypeptide was solubilized from the remaining paniculate fraction by 3 ml of 5 M guanidine HC1, followed by centrifugation at 30,000 X g for 30 min. Up to this point, all procedures had been carried out at 4°C and there have been five centrifugation steps for a total of two hours of spinning time (not including acceleration and deceleration time). Solubilized IL-lα polypeptide was purified by gel filtration chromatography on Sephacyl S-200 or Sephadex G-75, equilibrated and then eluted with 5 M guanidine HC1. This is an extremely cumbersome and difficult procedure even for a laboratory scale and would not be a feasible commercial scale procedure to isolate and purify IL-lα.

United States Patent 4,569,790 refers to a process to recover recombinant human interleukin-2 from transformed host E. coli cells. Host cells are first disrupted and then centrifuged to separate a soluble fraction from an insoluble fraction. Paniculate matter (insoluble fraction) was further washed to extract many of the soluble proteins. Interleukin-2 was located in the insoluble fraction. The insoluble fraction was treated with a chaotropic agent to selectively remove the bulk of the host E. coli proteins from the cellular debris by centrifugation. IL-2 polypeptide remained within the cellular debris. IL-2 was primarily hydrophobic at a reduced physiologic pH near its isoelectric point, and was primarily present as inclusion bodies of significant mass. Cellular debris and inclusion bodies were separated by selectively solubilizing the insoluble fraction with a neutral, aqueous buffer containing a reducing agent and a solubilizing agent (e.g. detergent or surfactant). IL-2 was organically extracted under reducing conditions with 2-butanol or 2-methyl-2-butanol to remove additional E. coli proteins or contaminants that have molecular weights close to that of IL-2. After the extraction was completed, the aqueous phase and the organic phase were separated. IL-2 was separated from the organic phase by acid precipitation.

Another procedure for extracting recombinant polypeptides from host cells is described in United States Patent 4,748,234. This multiple step procedure first disrupts the host cell membrane, removes salts from the disruptate by diafiltration with deionized water, redisrupts the desalted disruptate, increases the density or viscosity of the desalted disruptate, extracts the recombinant polypeptide by high-speed centrifugation, solubilizes the recombinant polypeptide under reducing conditions, organically extracts the solubilized recombinant polypeptide, and isolates the recombinant polypeptide from the organically extracted material. This eight step procedure utilizes high speed centrifugation, which is a difficult procedure with large, commercial scale volumes of material. This procedure further utilizes organic solvent extraction which results in hazardous waste disposal issues.

There is a need in the art to develop a commercial scale isolation and purification procedures for recombinant polypeptides that are expressed intracellularly. There is a further need in the an to be able to fractionate and purify recombinant polypeptides that are not excreted from host cells to provide compositions which are completely or substantially free of host cell nucleic acid and other host cell contaminating proteins such as bacterial cell proteins or endotoxins. There is a further need in the art to develop a commercial-scale process that can handle large volumes in a batch-type or a continuous procedure and that avoids the use of centrifugation or organic solvent extractions. This invention was made to address these needs.

SUMMARY OF THE INVENTION The present invention relates to a process for isolating a recombinant polypeptide accumulated intracellularly within a transformed host cell. The recombinant polypeptide (either soluble or insoluble) is isolated using a porous matrix to entrap host cells prior to disruption, or alternatively, to entrap cellular debris after disruption. To isolate a recombinant polypeptide produced intracellularly as a component of an insoluble fraction in a transformed host cell, the process of the invention comprises entrapping the host cells within a porous matrix; disrupting the host cell membrane to enable diffusion of soluble host cell proteins from the host cell; washing the entrapped host cell to remove the soluble host cell proteins while leaving the insoluble fraction within the porous matrix; and contacting the insoluble fraction within the porous matrix with a chaotropic agent to isolate the recombinant polypeptide from the insoluble fraction. Another process for isolating a recombinant polypeptide produced as an inclusion body in a transformed prokaryotic host cell comprises entrapping the host cells in an alginate porous matrix; disrupting the host cells with a chaotropic agent and a reducing agent and extracting soluble proteins from the porous matrix, wherein the recombinant polypeptide is solubilized by the chaotropic agent and reducing agent, and removed from the porous matrix in an extract. The recombinant polypeptide may further require refolding by diluting the extract with a refolding buffer, and purifying the recombinant polypeptide.

Yet another process for isolating a recombinant polypeptide produced as an inclusion body in a transformed prokaryotic host cell comprises disrupting the host cells to from a disruptate; entrapping non-soluble materials in the disruptate by forming a porous matrix; extracting the recombinant polypeptide from the porous matrix with an acid buffer at a pH from about 2.5 to about 4.0, whereby prokaryotic proteins and endotoxins precipitate and remain entrapped within the porous matrix; and purifying the extracted recombinant polypeptide.

Still another process for isolating a recombinant polypeptide produced as an inclusion body in a transformed prokaryotic host cell comprises disrupting the host cells to form a disruptate whereby the recombinant polypeptide is in a soluble component of the disruptate; entrapping insoluble components of the disruptate by forming a porous matrix; straining and washing the porous matrix from the soluble component of the disruptate; and purifying the recombinant polypeptide in the soluble component of the disruptate.

To isolate a recombinant polypeptide produced in a soluble fraction of a host cell, the process of the invention comprises disrupting the host cell in an acid pH to form a disruptate consisting of cellular debris, a soluble fraction containing the recombinant polypeptide, and precipitated host cell proteins precipitated by the acid environment; entraping the cellular debris and the precipitated host cell proteins in a porous matrix; separating the soluble fraction from the porous matrix; and recovering the recombinant polypeptide from the soluble fraction. This process uses a porous matrix to fractionate cellular debris and precipitated host cell proteins from the soluble recombinant polypeptide. Entrapment provides both a fractionation and purification step to remove cellular debris and host cell proteins precipitated at low pH.

The present invention further provides a method for isolating recombinant human IL-lβ from prokaryotic transformed host cells that produce IL-lβ, comprising the steps of: (1) disrupting the host cell membranes to form a disruptate; (2) acidifying the disruptate with an acid buffer at a pH from about 2.5 to about 4.0, whereby prokaryotic host cell proteins and endotoxins precipitate or aggregate into large insoluble masses; (3) entrapping insoluble materials in the acidified disruptate by forming a porous matrix; (4) removing soluble IL-lβ from the porous matrix; and (5) purifying IL-lβ. The present invention uses a porous matrix to separate soluble from insoluble materials, thus avoiding centrifugation. Further, the present invention utilizes an acid fraction step that can separate IL-lβ from host cell proteins and from endotoxins that tend to remain insoluble and precipitate under acid conditions.

Preferably, the porous matrix of each process of the present invention is formed with an alginate salt or another complex polysaccharide derived from a seaweed or algae source. Most preferably, the alginate porous matrix is formed from sodium alginate combined with calcium chloride to form porous alginate beads. Further, optimal acid pH conditions for entrapping host cell materials are from about pH 2.8 to about 3.2.

DETAILED DESCRIPTION OF THE INVENTION Each process of the present invention utilizes a porous matrix, preferably an alginate porous matrix, to entrap the host cells or insoluble debris. The use of a porous matrix allows large-scale isolation of recombinant polypeptides in a continuous manner, without having to resort to a more cumbersome batch process that utilizes centrifugation to separate soluble from insoluble materials. The use of a porous matrix further reduces endotoxin content of isolated recombinant polypeptide to reduce reliance upon purification processes to reduce endotoxin levels.

In one embodiment, a method for isolating insoluble recombinant polypeptides, such as interleukin-7 (IL-7), from prokaryotic transformed host cells comprises two steps. First, the cells are entrapped in a porous matrix. Second, recombinant polypeptide is extracted from the entrapped cells.

Preferably, prior to entrapping the cells, one concentrates the host cells within their fermentation broth. Concentration may be performed, for example, by means of diafiltration, filtration, centrifugation, or other conventional means. Concentration to an optical density of from about 40 to about 700, preferably from about 280 to about 600, at 550 nm, is preferred.

The entrapping step forms a porous matrix to entrap the prokaryotic host cells. Preferably, when forming an alginate porous matrix, one mixes a sodium alginate solution (or another alginate salt) with a suspension of the host cells or disruptate. Other complex polysaccharides that can form a porous matrix can be substituted in place of alginate.

Alginic acid is a co-polymer of β-D-mannuronic acid and α-L-glycuronic acid linked by the αl- 4 glycolytic linkages. Alginic acid, like carrageenan, is a constituent of marine algae. Alginic acid is produced by the brown algae of the phaeophyceae, which occur in intertidal zones and are free living. Phaeophyceae commonly are found in the Sargasso Sea. A primary commercial source of alginic acid is Macrocystis pyrifera which is a giant kelp harvested off the Pacific Coast of the United States. Alginic acid can also be obtained from Laminaria species which grow off the coast of Europe, Japan and Northeast America, and Ascophylum species which grow around the English coast. In nature, alginic acid occurs as its mixed salt with sodium, calcium and magnesium. Commercial preparation of alginic acid extracts alginic acid by digestion of the seaweed with sodium hydroxide to produce a dilute solution of sodium alginate. The dilute solution of sodium alginate can be filtered to remove particulate matter. Sodium alginate is then further purified. Alginate is commonly sold as a sodium salt. However, alginate is also commercially available as an ammonium or propylene glycol salt and as a propylene glycol ester of alginic acid. Sodium alginate is useful for forming a porous matrix because it has a high affinity for divalent cations, such as calcium, magnesium, barium, and strontium.

Various methods exist for entrapping host cells or debris and insoluble material within a disruptate within a porous matrix. For example, an alginate (e.g., sodium alginate) solution is mixed with the host cells to form a mixture. This mixture is slowly added to a salt solution comprising a divalent cation, preferably a calcium salt such as calcium chloride. The salt solution is gently stirred to allow newly formed porous matrix, in the form of beads, to move away from the entry point of the mixture of the two solutions.

In case of error the porous matrix is removed by adding a divalent cation chelating agent, such as EDTA or citrate, to remove the divalent cation from the porous matrix and thereby restore the alginate solution as a soluble suspension. The porous matrix is maintained by the presence of divalent cations.

The porosity of a calcium alginate porous matrix can be controlled by altering the starting concentration of alginate. Preferred concentrations of alginate in a host cell- alginate suspension is from about 1% to about 8% (w/v) alginate. A preferred concentration of a divalent cation salt (such as a calcium chloride) is from about 20 mM to about 200 mM salt. The porous matrix entraps host cells upon formation. Preferably, the porous matrix is formed in about 50 mM calcium. After the porous matrix is formed, it can be maintained in a lower concentration of divalent cation; for example, ten times lower than the concentration of divalent cation used in forming the porous matrix. If a monovalent cation is present, for example sodium, then the maintenance concentration of divalent cation should be increased. A second step of the inventive methods involve extracting insoluble recombinant polypeptide from the entrapped host cells within the porous matrix. This is accomplished by changing the buffer solution that bathes the entrapped host cells to an extraction buffer. The extraction buffer functions to disrupt entrapped host cells and possibly to solubilize the recombinant polypeptide. The extraction buffer comprises a chaotropic agent and , preferably, a reducing agent. Chaotropic agents include, for example, guanidine, urea, anionic surfactants, ammonia, sodium dodecyl sulfate (SDS), and combinations thereof. A preferred chaotropic agent is guanidine and a preferred concentration is from about 5 M to about 10 M. Reducing agents are selected from the group consisting of dithiothreital (DTT), glutathione, mercaptoethanol, lipoic acid, thioglycolic acid, thioredoxin, and combinations thereof. A preferred reducing agent is DTT at a concentration of from about 5 mM to about 20 mM. The extraction buffer may further comprise from about 1 mM to about 20 mM of a salt solution comprising a divalent cation. The presence of a divalent cation helps keep the porous matrix stable.

Extraction is usually performed at room temperature for at least about four hours. Preferably, the extraction step is performed twice, in an effort to reduce the volume of extract, and the extract, containing soluble recombinant polypeptide, is pooled for subsequent renaturation (if necessary) and purification. Another method for isolating a soluble recombinant polypeptide, such as human

IL-lβ, from prokaryotic transformed host cells comprises five steps. First, the host cell membranes are disrupted to form a disruptate. Various methods for host cell membrane disruption are available, including mechanical means, such as homogenization and sonication, and chemical means such as treatment with a chaotropic agent, under relatively mild conditions to avoid denaturation of recombinant polypeptide. Examples of suitable chaotropic agents include guanidine hydrochloride (less than 5 M) and various surfactants. Preferably, the host cell membrane is disrupted by physical means. Most preferably, the most cell membrane is disrupted by homogenization at about 8000 psi. Second, the disruptate containing a soluble recombinant polypeptide is acidified to acid conditions with a buffer. Under acid conditions (from about pH 2.5 to about pH 4.0) the recombinant polypeptide remains soluble, but many endogenous prokaryotic proteins, for example, endotoxins precipitate. At an acid pH, precipitated host cell proteins and endotoxins can be entrapped in a porous matrix formed in the next step. Preferred acid buffers include, for example, acetate, glycine HCl buffer and other buffers having a pH of from about 2.5 to about 4.0. The acid buffer further comprises a maintenance concentration of calcium or other divalent cation to enable formation of a porous matrix. Most preferably, the pH of the extraction buffer is from about 2.8 to about 3.2.

Third, a porous matrix is formed to entrap precipitated or insoluble materials in the disruptate. A porous matrix can be formed by forming a salt of a complex polysaccharide and a divalent cation. Examples of complex polysaccharide materials that can form a porous matrix include alginic acid, carrageenan, and other complex polysaccharides derived from marine materials (e.g., seaweed). Preferably, the porous matrix comprises alginate. A process for forming a porous matrix is described above. Soluble materials can be separated from the porous matrix, for example, by filtering or straining the solution. The porous matrix and entrapped insoluble materials are retained on the filter, while the filtrate contains soluble recombinant polypeptide. It is also possible, but not necessary, to use centrifugation to accomplish this separation. The recombinant polypeptide is then purified to further remove contaminating proteins and other materials. Common purification procedures, such as reverse phase high performance liquid chromatography (RP-HPLC) can be used. Suitable purification procedures for IL-lβ include, for example, the IL-lβ purification procedure described in U.S. Patent 4,801,686.

The advantage of using a porous matrix such as calcium alginate as an immobilization matrix (compared to low pH and removal by centrifugation or filtration) is that : (1) cell debris and precipitate is entrapped within the alginate porous matrix for easier removal; and (2) there is increased recombinant polypeptide purification achieved, and greater removal of host cell proteins, such as endotoxins, due to the ability of the porous matrix to entrap host cell proteins. The invention is illustrated by the following examples illustrating the use of an alginate porous matrix for isolating human IL-7 and human IL-lβ.

Example 1

This example illustrates a procedure for isolating human B -lβ from E. coli cells that produce recombinant IL-lβ. Approximately 1 liter of fermentation broth (optical density of 100 at 550 nm) containing E. coli cells expressing recombinant IL-lβ intracellularly in a soluble form is processed in a Gaulin homogenizer at about 8000 psi at temperatures of 4-10° C. Solid glycine is added to the solution to provide a final concentration of 0J M glycine at pH 2.8 (adjusted by addition of HCl). The resulting disruptate is allowed to stand overnight at about 4° C to allow host cell proteins and endotoxins to precipitate. Next, the cell disruptate is mixed with about 600 ml of 5% alginic acid and dripped into 3.2 liters of 0J M glycine HCl pH 2.8 containing 0J M calcium chloride. When the drops of cell disruptate/alginate mixture contact the extraction buffer, a skin of cross-linked calcium alginate porous matrix develops. The calcium alginate porous matrix entraps insoluble components, including larger molecular weight molecules, molecular aggregates, and precipitates. This procedure is carried out for a period of at least four hours. The resulting calcium alginate beads are separated from extraction buffer by filtration. Another 3.2 liters of extraction buffer is added for a second extraction of the calcium alginate porous matrix beads. Soluble IL- lβ from both extractions is pooled for purification.

IL-lβ extracted from the porous matrix is then purified by by batch cation exchange chromatography as follows. First, IL-lβ is equilibrated in SP-ToyoPearl® in 25 mM MES (2-(N-Moηpholine)- Ethanesulfonic acid) pH 5.0, and eluted from the cation exchanger in an increasing linear gradient of 0-0.4 M sodium chloride. The IL- lβ eluted from the cation exchanger is concentrated by diafiltration in a buffer of 10 mM Tris HCl pH 8.0. IL-lβ is applied to an anion exchange column (DEAE) and eluted from the column in equilibration buffer (10 mM Tris HCl pH 8.0) with an increasing linear gradient of 0-0.4M sodium chloride. IL-lβ is concentrated by diafiltration as before. IL-lβ is further fractionated in a Red Sepharose® column in equilibration buffer and eluted in 0 - 1.0M sodium chloride gradient. IL-lβ is dialyzed against a buffer of 0.2% sodium citrate pH 7.0, and 2% sodium chloride for a preparation of purified II- lβ.

Example 2 This example illustrates an isolation, renaturation and purification of human IL- 7 from transformed E. coli host cells. IL-7 was produced in the form of inclusion bodies by incubating transformed E. coli host cells in a fermentation broth. A 14 liter fermentation broth was concentrated to 2 liters by a diafiltration method. Diafiltration was accomplished by pumping the fermentation broth through a 0.45 μm filter in a tangential flow. Buffer exchange was performed 10 times by adding 2 liters of 20 mM Tris pH 8.0 and concentrated to 2 liters. The concentrated cells were entrapped in an alginate porous matrix. The alginate porous matrix was formed with a 5% (w/w) solution of alginic acid (Sigma, St. Louis, MO) dissolved in deionized water the day before it was used. Approximately 1.4 liters of 5% alginic acid was mixed with 2 liters of concentrated fermentation solution to form a homogenous slurry. The slurry was pumped through a multi-nozzle immobilization device placed approximately 15-25 cm above a calcium solution, such that droplets formed (approximately 2-3 mm diameter) when entering into a tank. The tank contained 8 liters of 50 mM calcium chloride in 50 mM Tris HCl buffer pH 8.0. No stirring of this solution was necessary. The flow rate was chosen such that droplets were formed without sticking to each other when entering into the calcium chloride solution. The alginate porous matrix formed in the tank and entrapped host cells. The alginate porous matrix was incubated for approximately one hour at room temperature. The alginate porous matrix was then washed at least two times with about 6 liters of 5 mM calcium chloride in 0J M Tris HCl buffer to remove cells and other fermentation debris that did not become entrapped in the porous matrix. The alginate porous matrix was separated from this solution with a strainer. The extraction step was performed twice with 8 liters of 7 M guanidine hydrochloride in 0J M Tris HCl, 5 mM calcium chloride and 10 mM of the reducing agent, DTT for each extraction of IL-7 from the alginate porous matrix. Both extraction steps required at least about 6 hours of incubation at room temperature.

Renaturing was accomplished by diluting the extract containing IL-7 to a theoretical optical density of approximately 7 at 550 nm. This corresponds to a final protein concentration of approximately 0.2 mg ml in approximately 2 M guanidine. The extract was diluted with a refolding buffer comprising 0.2 mM glutathione (oxidized), 1 mM glutathione (reduced), 2 mM of a chelating agent (EDTA), 0J M ammonium sulfate and 0J M Tris at a pH of 8.0. The volume of extracted IL-7 in refolding buffer was approximately 120 to 180 liters. The refolding buffer was stirred in a large tank with an open lid at room temperature to allow slow air oxidation. The tank lid was open to allow for a mildly oxidizing atmosphere.

Purification was a multi-step process that began by diafiltering IL-7. Approximately 20 liters of refolding buffer was diafiltered against approximately 100 liters of purification buffer. Purification buffer comprised 50 mM ammonium sulfate and 50 mM Tris at a pH of 7.4. Diafiltration was performed continuously with a slow decrease of refolding buffer concentration.

After diafiltration the solution was refrigerated at approximately 4° C overnight. This allowed for precipitation of denatured proteins. The precipitated proteins were removed by filtering the solution through a 0.2 μ filter.

Next, the IL-7 solution and purification buffer was purified by 3 column chromatography steps. First, the solution was applied to a cation exchange (Mono S 60/100, Pharmacia) column. Approximately 40 liters of the IL-7 solution was applied to the column at a rate of approximately 25 ml/min. Application of this solution to the column was followed by 10 column volumes of wash buffer (purification buffer) also applied at a flow rate of 25 ml/min. The column was eluted with 15 column volumes of a linear salt gradient at a flow rate of 50 ml min. The salt gradient ranged from 0-1.0 M NaCl. IL-7 eluted between 5.6% and 24.4% of the second buffer, which corresponds to 0.07 M NaCl to 0.24 M NaCl. Each fraction within the range contained IL-7 and was pooled for further purification.

After each use of the cation exchange column, the column was washed. We followed a washing procedure adding 1/2 column volume of filtered 2 M NaCl solution at a flow rate of 50 cm/hour (24 ml/min), followed by 1 column volume of water and 4 column volumes of 1 M sodium hydroxide at 40 cm/hour (19 ml min). This was followed sequentially by 1 column volume of water, 2 column volumes of 75% acetic acid, 2 column volumes of low ionic strength buffer and 1 column volume of 2 M NaCl at a flow rate of 100 cm/hour (48 ml/min). Lastly, 10 column volumes of 40% ethanol was added at a flow rate of 40 cm/hour (19 ml/min). This procedure was sufficient to regenerate the column for further purification uses.

The pooled IL-7 solution fractions were added to a C18 (Vydac®) column, whose dimensions were 2.5 cm x 10 cm in a 0.1% trifluoracetic acid (TFA) buffer with acetonitrile elution. The column was first equilibrated in a 0.1% TFA solution. The flow rate was 20 ml/min. Elution was with a linear gradient of acetonitrile in 0.1% TFA over 20 column volumes. IL-7 eluted at 60-65% acetonitrile.

RP-HPLC fractions containing IL-7 were pooled and applied to a Mono-S cation exchange column (60/100) in buffer A (50 mM sodium acetate pH 4.7) and eluted with a Unear gradient of (0-100%) buffer B (1 M NaCl in 50 mM sodium acetate pH 4.7). After application of the IL-7 pooled fraction, the column was washed with 10 column volumes of 50 mM sodium acetate (pH 4.7), and eluted 15 column volumes of with buffer B. Substantially homogenous IL-7 eluted at 54%-58% buffer B.

The procedure employing an alginate porous matrix produced purified hIL-7 with an endotoxin level less than 0.012 ng/mg IL-7, which is the limit of detection. Thus, no endotoxin could be detected.

Claims

CLAIMSWhat is claimed is:

1. A process for isolating a recombinant polypeptide produced as an inclusion body in a transformed prokaryotic host cell, comprising:

(a) entrapping the host cells in a porous matrix;

(b) disrupting the host cells to enable diffusion and removal of host cell proteins from the porous matrix;

(c) contacting the porous matrix with a chaotropic agent to solubilize the recombinant polypeptide; and

(d) removing the soluble recombinant polypeptide from the porous matrix.

2. A process for isolating and purifying a recombinant polypeptide produced in a transformed prokaryotic host cell, comprising:

(a) entrapping the host cells in a porous matrix;

(b) disrupting the host cells with a chaotropic agent and a reducing agent and extracting soluble proteins from the porous matrix, wherein the recombinant polypeptide is solubilized by the chaotropic agent and reducing agent, and removed from the porous matrix in an extract;

(c) refolding the recombinant polypeptide by diluting the extract with a refolding buffer, and (d) purifying the recombinant polypeptide.

3. A process for isolating a recombinant polypeptide produced in a transformed prokaryotic host cell, comprising:

(a) disrupting the host cells to form a disruptate; (b) entrapping non-soluble materials in the disruptate by forming an alginate porous matrix;

(c) extracting the recombinant polypeptide from the porous matrix with an acid buffer at a pH from about 2.5 to about 4.0, whereby prokaryotic proteins and endotoxins precipitate and remain entrapped within the porous matrix; and (d) purifying the extracted recombinant polypeptide.

4. A process for isolating a recombinant polypeptide produced in a transformed prokaryotic host cell, comprising:

(a) disrupting the host cells to form a disruptate whereby the recombinant polypeptide is soluble component of the disruptate; (b) entrapping insoluble components of the disruptate by forming an alginate matrix;

(c) straining and washing the alginate porous matrix from the soluble component of the disruptate; and

(d) purifying the recombinant polypeptide in the soluble component of the disruptate.

5. The process of claim 1 wherein the alginate porous matrix is formed by mixing a 2.0% to 4.0% (w/v) solution of sodium alginate with the disruptate or a host cell suspension adding this mixture to a 20 to 100 mM calcium salt solution, and allowing the alginate porous matrix to form.

6. The process of claim 2 wherein the alginate porous matrix is formed by mixing a 2.0% to 4.0% (w/v) solution of sodium alginate with the disruptate or a host cell suspension adding this mixture to a 20 to 100 mM calcium salt solution, and allowing the alginate porous matrix to form.

7. The process of claim 3 wherein the alginate porous matrix is formed by mixing a 2.0% to 4.0% (w/v) solution of sodium alginate with the disruptate or a host cell suspension adding this mixture to a 20 to 100 mM calcium salt solution, and allowing the alginate porous matrix to form.

8. The process of claim 4 wherein the alginate porous matrix is formed by mixing a 2.0% to 4.0% (w/v) solution of sodium alginate with the disruptate or a host cell suspension adding this mixture to a 20 to 100 mM calcium salt solution, and allowing the alginate porous matrix to form.

9. A method of using a porous matrix in a process for isolating a recombinant polypeptide wherein the porous matrix binds insoluble material and facilitates separation of insoluble material from soluble material.

10. A method for isolating recombinant Interleukin- 1 β (IL- 1 β) from prokaryotic transformed host cells that produce IL-lβ, comprising: (a) disrupting the host cell membranes to form a disruptate;

(b) acidifying the disruptate with an acid buffer at a pH from about 2.5 to about 4.0, whereby prokaryotic proteins and endotoxins precipitate;

(c) entrapping insoluble materials in the acidified disruptate by forming a porous matrix;

(d) separating soluble IL-lβ from the porous matrix; and

(e) purifying soluble IL-lβ.

11. The method of claim 10 wherein cell membrane disruption is caused by a technique selected from the group consisting of homogenization, sonication, and treatment with a chaotropic agent.

12. The method of claim 11 wherein cell membrane disruption technique is homogenization at about 8000 psi.

13. The method of claim 10 wherein the porous matrix is formed by mixing a 2.0% to 4.0% (w/v) solution of sodium alginate with the acidified disruptate, adding this mixture to a 2.0 to 100 mM calcium salt solution, and allowing the alginate porous matrix to form.

14. The method of claim 10 wherein the acid buffer comprises an acetate buffer or a glycine/HCl buffer and the pH conditions are from about 2.8 to about 3.2.

15. The method of claim 10 wherein soluble IL- 1 β is purified by a high performance liquid chromatography procedure.