WO2002032537A2

WO2002032537A2 - Improved method of expanded bed chromatography

Info

Publication number: WO2002032537A2
Application number: PCT/US2001/027736
Authority: WO
Inventors: Walter Francis Junior Prouty
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2000-10-13
Filing date: 2001-09-28
Publication date: 2002-04-25
Anticipated expiration: 2003-04-13
Also published as: WO2002032537A3; AU2001296228A1

Abstract

The present invention is a method of separating a desired component (e.g., a biomolecule such as a recombinant protein) from a mixture such as an unclarified feedstock. In a first step, the mixture is solubilized in an aqueous urea solution to form a sample solution. In a second step, the sample solution is passed upwards through a bed of resin, thereby binding the desired component to the resin and separating the desired component from the mixture. The sample solution comprises a sufficiently high concentration of urea to reduce fouling of resin when the sample solution is passed through the resin.

Description

IMPROVED METHOD OP EXPANDED BED CHROMATOGRAPHY

Expanded Bed Chromatography (EBC) is a method for recovering biomolecules directly from unclarified feedstocks such as fermentation broths or cell homogenates. EBC is described in U.S. Patent No. 5,522,993 to Carlsson, et al . and Barnfield, et al . , Bioprocess Engineering 16: 51 (1997), the entire teachings of which are incorporated herein by reference.

EBC is a type of chromatography in which liquid is passed upwards through a bed of absorptive resin. The resin particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward flow, thereby forming a stabilized bed that produces chromatographic plates. While it operates much as a packed chromatographic bed, the upward flow of liquid allows the bed to expand and create space between the suspended resin particles. Thus, it is possible to pass a stream of material through the column that contains substantial amounts of particulate matter. An important advantage of this technology over downstream purification is that pre- filtration of the material going over the expanded bed is not required. Therefore, the process may be viewed as combining two steps (filtration and chromatographic capture) into one (EBC) . The process is thus more economical than packed bed chromatography when isolating biomolecules from solutions that contain both soluble and insoluble impurities . Unclarified feedstocks contain high levels of impurities which can rapidly damage or "foul" resins used in EBC. Fouling manifests itself as visible resin clumping in the expanded mode, which is analogous to channels in a packed bed. As a consequence, charging material does not contact the clumped portion of the resin, thereby reducing the binding efficiency of the column. The clumps can grow to the extent where fluid is visibly streaming through wide channels. Typically, the resin must be regenerated or replaced after about five to seven runs when EBC is used to remove solutes from unclarified feedstocks. Regeneration of the resin, which must often be done out of column, is both costly and inefficient. The cost effectiveness of EBC in industrial processes is dependent upon the development of efficient methods for extending the longevity of the resins used in EBC.

It has now been found that the lifetime of resin particles used in Expanded Bed Chromatography (EBC) to recover biomolecules from unclarified feedstocks can be significantly prolonged when the charge and wash solution contain a sufficient quantity of urea. Specifically, resin lifetime was extended to at least fifty runs and beyond with column charges and wash solutions containing 6.3 M urea (Example 3) . In contrast, replacement of the resin or harsh out-of-column cleaning procedures were required after only about five to seven runs when urea was not used. Based on this discovery, methods of isolating biomolecules from unclarified feedstocks by EBC while minimizing column fouling are disclosed herein. The present invention is a method of separating a desired component (e.g., a biomolecule such as a recombinant protein) from a mixture such as an unclarified feedstock. The mixture is solubilized in an aqueous urea solution to form a sample solution. The sample solution is then passed upwards through a bed of resin, thereby binding the desired component to the resin and separating the desired component from the mixture. The sample solution comprises a sufficiently high concentration of urea to reduce fouling of resin when the sample solution is passed through the resin. Preferably, an aqueous urea solution is then passed upwards through the column to elute undesired soluble and insoluble components. The wash solution also contains a sufficient amount of urea to reduce fouling of the resin. The method of the present invention utilizes urea to reduce resin fouling in EBC. The use of urea allows multiple runs with unclarified feedstocks (fifty runs and greater) without the need to change or regenerate the resin. Reduction in fouling is independent of column size. Thus, this improvement can be used when EBC is carried out on an industrial scale, for example, with 40 liter columns. Urea does not raise the solution conductivity or compete with solute for binding sites on the resin and therefore its use does not affect the yield of the recovered desired product.

The present invention is directed to an improved method of performing expanded bed chromatography (EBC) . The improvement comprises maintaining a sufficiently high concentration of urea in the EBC column charge and wash solution to reduce or substantially prevent fouling of the resin during the chromatography.

EBC is a purification procedure that is well known in the art and is described in detail in US Patent No. 5,522,993 to Carlsson, et al . and and Barnfield, et al . , Bioprocess Engineering 16: 57 (1997) . Specifically, EBC can be used to separate a desired soluble component from an impure mixture, which can include insoluble particulate components. Like conventional packed bed chromatography, EBC employs a column which contains a bed of solid phase polymer, also referred to as a "resin" . The resin selectively binds the desired component over other impurities. EBC also works by differential binding, e.g., some bound contaminants can be eluted selectively by a wash step, leaving the desired component bound to the resin for later elution. Alternatively, an elution step can selectively elute the desired component while leaving more tightly bound contaminants on the resin.

EBC can be used when a suitable solid phase resin is available that selectively or differentially binds the desired component over other impurity (ies) present in the mixture .

EBC differs from conventional packed bed chromatography in that the chromatography is run in an "upflow manner", i.e., the column charge and wash solutions are introduced or loaded sequentially onto the column from the bottom and are then pumped upwards through the column. The desired component binds to the resin, while the impurities pass upwards and out of the top of the column. The desired component can then be eluted from the resin by passing a suitable elution solution through the column in either a downflow or upflow manner.

The tendency of the resin particles to sediment is balanced against the pressure of fluids moving upwards through the column. As a result, resin particles are suspended during EBC. Insoluble, particulate matter can pass between the suspended resin particles . EBC therefore has the effect of combining a chromatography step and filtration step and can separate a desired component from both particulate matter and soluble impurities. As a consequence, EBC is particularly well suited for purifying biomolecules from unclarified feedstocks, e.g., eukaryotic or prokaryotic cell extracts, cell lysates or fermentation broths from, mammalian, fungal, plant or yeast cells. EBC is therefore often used to separate recombinant proteins from insoluble cellular debris and soluble biomolecules (e.g., lipids, salts, amino acids, sugars, proteins and nucleic acids) in the cell homongenates or fermentation broths in which they are produced. EBC has been described for ion exchange chromatography (anionic or cationic) as well as hydrophobic interaction, metal chelating and Protein A adsorption modes. The method can be used to purify other biomolecules which adhere to the resin, including nucleic acids, carbohydrates and glycoproteins and the like.

To carry out EBC, the mixture from which the desired component is to be isolated is solubilized to form a column charge. As used herein, the term "solubilize" refers to combining the mixture with an aqueous solvent or a suitable organic/aqueous solvent mixture so that the desired component dissolves. Optionally, the column charge can contain other components such as detergents (non-ionic detergents, if ion exchange chromatography is being used) . Some or all of the other components in the mixture may also dissolve. Thus, solubilizing the mixture results in a solution that may contain some particulate matter. The resulting solution, also referred to as the "column charge" or "sample solution", is used to load or apply the mixture from which a desired component is to be isolated to a chromatography column; in the case of EBC, the column is loaded from the bottom. The concentration of the desired component in the column charge is chosen such that the desired component binds to the resin in the column and is typically between about 0.01 mg/mL and about 10.0 mg/mL. The wash solution is loaded onto the column immediately after the column charge to elute both the soluble and insoluble impurities from the column. As the wash solution passes upward through the column, insoluble components and soluble components which bind to the resin less strongly than the desired component pass through the column and out the top, leaving the desired component bound to the resin. The wash solution and the column charge are typically the same solution, but for the presence of the impure mixture in the column charge. Optionally, however, the wash solution can differ from the column charge, provided that the desired component remains bound to the resin as the wash solution is being passed through the column. When ion exchange chromatography is being used, for example, the wash solution may contain salts or have a different pH such that the desired component remains bound to the resin while contaminants are selectively eluted.

In the method of the present invention, the column charge and wash solution also contain a sufficiently large concentration of urea to reduce or substantially prevent column fouling. Column fouling refers to reduced affinity of the resin for components which are normally absorbed onto the resin, increased clumping of the resin and increased channeling in the column. Column fouling results in changes in fluid dynamics wherein the purification of the desired component is adversely effected. When fouling reaches a certain level, the resin must be regenerated or replaced. Maintaining the urea concentration above about 5.0 M preferably above about 6.0 M and preferably between about 6.0 M and about 8.0 M, more preferably between about 5.0 M and about 7.0 M, even more preferably between about 6.3 M and about 7.0 M in these solutions increases the longevity of the resin to greater than about 25 runs when the resin is used to isolate a recombinant protein or peptide from an unclarified solution. Although lesser concentrations of urea can also be used, the increase in resin longevity is not as great. The concentration of urea is sufficient to reduce fouling when the resin lifetime is longer in the presence of urea than in its absence. Column lifetime can be measured, for example, by the number of runs before the resin needs to be regenerated or replaced.

The concentration of urea in the column charge and wash solution can be the same or different, but is preferably the same or higher in the wash solution. Most preferably the concentration of urea in the wash solution is higher than that used in the column charge. The higher urea concentration in the wash creates separate layers wherein there is very little mixing between the wash solution and the column charge. The concentration differential works to create a meniscus or plug that allows particulate matter to be pushed out of the top of the column much more efficiently. Preferably the column charge has a concentration of urea between about 5 M and 6.5 M and the wash has a urea concentration greater than about 6.5 M. Preferably the urea concentration in the wash is between about 7.0 M and about 8.0 M.

Preferably, wash solution is passed through the column until substantially all of the non-bound components (soluble and insoluble) have eluted from the column. Thus, the use of wash solution is continued until the eluent is clear and colorless. Alternatively, the refractive index or visible/ultraviolet absorption of the eluent can be monitored to determine when the non-bound components have completely washed off the column.

EBC requires that the bed of resin be stable and fluidized. A "stable, fluidized bed" refers to resin beds in which there is little translational movement of the individual resin particles. Resin particles are suspended in equilibrium due to the balance between particle sedimentation velocity and upward flow. Thus, a given resin particle will remain within a limited volume that is a minute fraction of the total bed volume during the chromatography. In addition, there is little or no channeling.

The flow rate during EBC is selected so that a stable, fluidized bed is maintained. Although suitable rates may vary according to the type of resin, charge solution density and column size and shape, one of ordinary skill in the art can select flow rates using routine experimentation. The flow rate should not be so great that the bed expands and contacts the top adapter or top of the column. Flow rates from between about 5-3000 cm/hour are known in the art, typically between about 5-500 cm/hour and more typically between about 50-200 cm/hour.

The resins are selected so that a stable, fluidized bed is maintained. To maintain a stable, fluidized body, the resin particles should be relatively small to allow short diffusion distances and have a high density. For example, resin particles with a diameter between about 100-1000 jura and a particle density between about 1.10-1.50 g/ml when hydrated are typical, although more dense particles are known in the art (e.g., zirconium-based particles). The particles can be spherical or irregular in shape.

The resin particles typically comprise a polymer matrix into which glass, quartz, silica particles or zirconium are incorporated and should be large enough so as not to pass through the screen at the bottom of the column. For example, spherical and irregular shaped glass and silica particles having a size in the range of from 100-300 jura are typical, although smaller particles are also known in the art. The incorporated material is typically in the range of from about 5-50% of the weight of the wet final particle. The polymer is derivatized to selectively bind the desired component. Examples of suitable polymers include mono- or polyvinyl monomers such as acrylates, methacrylates or vinylbenzenes, or naturally-occurring polymers such as polysaccharides (e.g., agarose, starch, cellulose or derivatives thereof) . Optionally, the polymer is crosslinked for the desired rigidity and pore distribution. Ion exchange resins contain groups which are suitable for binding ions, e.g., diethylamino ethyl or quarternary amine groups.

Suitable resin particles are available commercially from Amersha Pharmacia Biotech under the tradename STREAMLINE. Examples are found in the 2000 Pharmacia Biotech Catalog on pages 162-63, and include STREAMLINE CHELATING, Catolog No. 17-1280 (a metal chelating resin),

STREAMLINE DEAE, Catolog No. 17-0994 (a weak anion exchange resin), STREAMLINE HEPARIN, Catalog No. 17-1284 (a polysaccharide anticoagulant resin) , STREAMLINE PHENYL, Catalog 17-5121 (a hydrophobic interaction resin) , STREAMLINE QXL, Catolog No. 17-5075 (a strong, highly substituted anion exchange resin) , STREAMLINE RPROTEINA, Catalog No. 17-1281 (an immunoglobulin binding), STREAMLINE SP, Catalog No. 17-0993 (a strong cation exchange resin) and STREAMLINE SPXL 17-5076 (a highly substituted cation exchange resin) . All of these resins are macroporous cross- linked 6% agarose containing a crystalline quartz core with

100-300 μm spherical particles. Ion exchange resins (anionic or cationic) are preferably used to separate recombinant proteins from unclarified feedstocks. The precise amount of resin that is used can be readily determined by one of ordinary skill in the art, although there may be some variation depending upon the application and the feedstock. Typically, between about 10 to about 50 grams of crude product can be loaded on one liter of resin, more typically between about 20 to about 30 grams.

After the insoluble debris and soluble contaminants have passed through the column, the desired component is desorbed from the column with an elution buffer. The elution buffer can be applied in an upflow fashion as with the column charge and wash solution. Alternatively and preferably, the elution buffer is applied in a downflow fashion; the resin particles settle and the elution buffer is loaded from the top of the column as in conventional packed bed chromatography. Downflow elution has the advantage in that no movement of the top adapter is required, i.e., a fixed volume column can be used. This provides for greater ease of operation and the ability to use a less expensive, fixed head column. Use of urea in the charge and wash and lower density nonurea-containing elution buffer allows the use of downflow with a fixed head column. This provides an operationally easy mode of column operation and is a major advantage of the procedure described herein. An "elution buffer" is a solution which, when passed through a chromatography column, results in decreased binding (or desorption) between a resin and a component that is bound to the resin. For ion exchange resins, elution buffers typically contain ionic components which compete with the desired component for the binding sites on the resin. Salt solutions such as aqueous sodium chloride, potassium chloride and other physiologically acceptable salts are examples of suitable elution buffers for ion exchange resins. Typically, salt concentrations of between about 0.3 M and about 1.0 M are used in the elution buffer. Changes in the pH can also change the binding affinity of the desired component to the resin.

The elution buffer is generally less dense than the wash solution, which contains urea. Thus, the more dense wash solution will move down the column with a sharp band of the water elution buffer on top of it, resulting in "plug" flow. Plug flow results in excellent chromatographic performance by providing a sharp elution band with little or no band spreading at the urea/water interface. In addition, plug flow elutes the bound material in a smaller volume than would be attained in an upward flow mode, which is an asset to downstream processing. Water is also used as an elution buffer with hydrophobic interaction chromatography, where high salt drives solutes onto the resin. After elution, the desired component can be further purified or isolated by any suitable means such as lyophilization, spray drying or crystallization.

The invention described herein is preferably used as an early purification step in a series of purification steps that may be necessary to purify a particular peptide or protein to homogeneity such that the protein can be formulated and administered as a pharmaceutically acceptable drug product. Preferably the purification step described herein is used as a first step to purify particulate matter from a solubilized suspension of prokaryotic or eukaryotic cells that have been engineered to express the peptide or protein of interest.

A pharmaceutically acceptable drug product may have the active protein or peptide combined with a pharmaceutically- acceptable buffer, and the pH adjusted to provide acceptable stability and solubility properties, and a pH acceptable for parenteral administration. Pharmaceutically-acceptable anti-microbial agents may also be added. Meta-cresol and phenol are preferred pharmaceutically-acceptable antimicrobial agents. One or more pharmaceutically-acceptable salts may also be added to adjust the ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin is an example of an isotonicity-adjusting excipient.

"Pharmaceutically acceptable" means suitable for administration to a human, and does not contain toxic elements, undesirable contaminants or the like, and does not interfere with the activity of the active compounds therein.

The present invention is suitable for use with any protein or peptide that is not irreversibly denatured when solubilized in the presence of the concentrations of urea described herein. Most biologically active proteins and peptides are not overly sensitive to urea because urea typically does not cause breaks in disulfide bonds. Thus, proteins or peptides that do unfold or change conformation in the presence of urea can generally be renatured by diluting with elution buffer that does not contain urea.

Examples of proteins and peptides suitable for use in the processes of the present invention include: Glucagon-like peptide-1 (GLP-1) and analogs thereof such as Val⁸-GLP-l(7-37)OH or Arg³⁴-GLP-1 (7-37) OH. For example, U.S. Patent No. 5,118,666; U.S. Patent No 5,977,071; U.S. Patent No. 5,545,618; U.S. Patent No. 5,705,483; U.S. Patent No . 5,977,071; U.S. Patent No. 6,133,235; and Adelhorst, et al., ^". Biol . Chem . 269 : 6275 (1994) disclose GLP-1 analogs that can be purified using the invention described herein. Additional examples of proteins and peptides useful in the processes of the present invention include exendin-3, exendin-4, analogs of exendin-3 and exendin-4, human insulin, human insulin analogs such as LysB27ProB28-human insulin or AspB28-insulin, human growth human and analogs thereof, parathyroid hormone and analogs thereof.

Other suitable peptides and proteins (including glycosylated and nonglycosylated proteins and cytokines) include but are not limited to, calcitonin, erythropoietin (EPO) , factor IX, factor VIII, 5-lipoxygenase and cyclooxygenase products and inhibitors, granulocyte colony stimulating factor (G-CSF) , granulocyte macrophage colony stimulating factor (GM-CSF) , macrophage colony stimulating factor (M-CSF) , nerve growth factor (NGF) , ciliary neurotrophic factor (CNF) , defensins, chemokines, growth hormone releasing factor (GRF), insulinlike growth factor (IGF-1) , growth hormone, heparins (regular and low molecular weight) , cyclosporin, insulin, leptin and its analogs and inhibitors interferon- .alpha. , interferon- .beta. , interferon- .gamma. , interleukins (e.g. interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6) , interleukin-11, interleukin- 12), interleukin-1 receptor antagonist, interleukin-1 receptor (IL-1R) , luteinizing hormone releasing hormone (LHRH) agonists and antagonists, nafarelin, goserelin, leuprolide, somatostatin analog (e.g. octreotide) , vasopressin analogs, peptide Y, gastrins, CCK peptides, thymosin- .alpha. -1, Ilb/IIIa inhibitors, .alpha. -1 antitrypsin, anti-RSV antibody, cystic fibrosis transmembrane regulator (CFTR) gene, integrins, selectins, deoxyribonuclease (DNase) , and FSH

The peptide or protein of interest may be expressed as a fusion protein wherein this fusion protein contains a cleavable component such as a carboxypeptidase leader sequence fused to the active protein or peptide. Once purified from prokaryotic or eurkaryotic cellular debris, fusion proteins can be further purified and cleaved such that the active protein or peptide is released. The active component then can be formulated such that is suitable as a pharmaceutically acceptable drug product. The columns used for EBC typically have: a) a distributor in form of a bottom port that distributes an inlet flow- of liquid equally over the cross-sectional area of the column, (b) a top port, and (c) there between a void volume for holding resin particles. Columns suitable for use in EBC are commercially available from Pharmacia Biotech under the tradename STREAMLINE (see page 94 of the 2000 Pharmacia Biotech catalog), e.g., STREAMLINE 50, STREAMLINE 100, STREAMLINE 200 and STREAMLINE CD. Other columns are available from Upfront Chromatography A/S, Copenhagen, Denmark.

A system for carrying out EBC generally includes pumps for supplying the appropriate solutions, valves, as appropriate, to switch the flow of solvents into the column, a sample container, a column as described above, an adjustable upper adapter and a fraction collector. The column is loaded with a resin suitable for selectively binding the desired component. The wash solution is then pumped into the bottom of column with the outflow at the top of the column connected to waste. Next, the column charge is pumped into the column under conditions so that a stabilized fluidized bed is maintained. When the charge has been transferred to the column, a wash solution is pumped into the column. After the fluidized bed has been thoroughly washed, and unbound components such as cell particles have been eluted to waste through the top of the column, the first phase of the procedure is finished.

The flow through the column is reversed for elution of the bound sample components. The valves are switched so that the flow is from a pump through the top of the column out of the bottom of the column to the fraction collector. After the bed has been allowed to settle, and optionally further converted to a packed bed by moving the upper adapter down to the bed surface, a suitable elution solution is introduced via pump into the bed whereby the desired component is released and collected. The upper adapter, which is of the traditional type found in a great number of columns for chromatography, is adjustable to a desired position in the column and contains a net to prevent beads from passing out the top of the column.

Alternatively, elution of the column is carried out in the fluidized mode by introducing the elution solution via a pump into the bottom of the column and collecting the sample constituents released from the resin with a fraction collector connected to the upper outlet of the column. In this case there is no need for a movable upper adapter in the column.

Example 1 - Preparation of the Column Charge

E. coli cells that were genetically engineered to express a fusion protein comprised of a carboxypeptidase leader sequence fused to the GLP-1 analog, Val⁸-GLP-1 (7-37 ) OH (a thirty-one amino acid peptide) were diluted with one volume of water and collected by centrifugation on a disk stack centrifuge. This cell paste containing the engineered fusion protein could be frozen for later use. To extract the fusion protein, the E. coli cell paste was suspended at up to 8% solids in 7 M urea containing 10 mM ethylendiamine, 0.5 M EDTA at pH 8.0. The pH of the mixture was raised to 11.5 using 10% NaOH. The mixture was incubated for 40 to 60 minutes at ambient temperature and then brought back to pH 8.0 using 10% HCl . Due to the volume of the cell paste, the concentration of the urea was between 5.5 and 6.3 M.

Example 2 - Purification by Expanded Bed Chromatography

The column charge was prepared from extracted fusion protein as in Example 1 by dilution to 4% solids with 7 M urea (final volume 450 ml) containing 5.32 g fusion protein/ml. The column tube (2.5 x 100 cm STREAMLINE column) was filled with 75 milliliters of STREAMLINE DEAE resin. The resin and column were treated sequentially according to the following table in the order as shown:

Note that all operations are with upflow, except in the elution step. In this column run, the column top adaptor was not moved, i.e. the column was operated in fixed head and fixed volume mode. The last step, washing with ethanol/acetic acid, was found to be unnecessary for every run. Rather, good performance was observed when it was utilized every fifth run.

Results of product distribution are shown in the following table:

Accountability: 101.5%

The resin used in this example had been used in 13 previous cycles with the conditions described above. Some of the fusion protein had eluted during the charge and wash, suggesting that resin was slightly overloaded at 31.9 milligrams of fusion per milliliter of packed resin. Mainstream pool was made by including all effluent that had ultraviolet absorption above 9 optical density units during the running of the aqueous, non-urea containing elution buffer. Product purity as measured by high performance liquid chromatography improved from about 20% pure in the charge solution to about 80% or better purity in the mainstream pool .

EXAMPLE 3. Repetitive runs in lab scale column

This example was run to test the effect of repetitive runs on the performances of the EBC STREAMLINE DEAE column using starting material as prepared in Example 1 and the protocol essentially as indicated Example 2. Centrifuged cell paste (250 milliliters) was diluted with extraction buffer (7.0 M Urea, 20 mM Ethylene Diamine, 845 milliliters), pH adjusted to 11.5 for about 60 minutes, then back to 8.0. Finally, the charge material was diluted with additional urea (7.0 M, 1300 milliliters) to reduce conductivity to about 3 milliSiemens and bring urea concentration to about 6.3 M. The final ethanol and acetic acid washes were run after runs 7, 29 and 50. What is shown below is comparative data from runs number 5, 30 and 50. In each run, 30 to 40 grams of fusion protein were charged per Liter of settled resin volume. All runs were made on a STREAMLINE 25 column with the top adaptor fixed in place. STREAMLINE DEAE (76 milliliters) was used in the column and bed expansion during the upflow portion of the runs was maintained at 2.5 to 2.7 fold by adjustment of flow rates.

Example 4. Pilot plant scale of EBC

Fusion protein charge was prepared as in Example 1. The column was a STREAMLINE 200 and was filled with about 10 Liters if STREAMLINE DEAE resin, i.e., about 32 centimeter bed height. Six successive runs were made on the column using running positions, buffers and relative buffer volumes as described in Example 2. Results of runs 4 and 6 are shown in the data box below to demonstrate reproducibility and scaleability of the process from a 2.5 centimeter diameter column to a 20 centimeter diameter column. The ethanol wash described as the last step in Example 2 was not run in between any of these 6 column runs .

Run 4:

Run 5:

Example 5. Scale up to production scale column run

The charge solution was prepared essentially as described in Example 1. The fusion protein solution was charged onto a STREAMLINE 400 column filled with about 40 Liters of STREAMLINE DEAE resin (40 x 32 cm of packed resin) . Again, buffers as described in Example 2 were run in the same order for this scaled up version of the EBC column run. The results of the run are described in Example 3.

Mainpeak pool was 82% pure as measured by high performance liquid chromatography.

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMSWhat is claimed:

1. A method of separating a desired component from a mixture, said method comprising the steps of: a) solubilizing the mixture in an aqueous urea solution to form a sample solution; b) passing the sample solution upwards through a bed of resin having affinity for the desired component, thereby binding the desired component to the resin and separating the desired component from the mixture, wherein said sample solution comprises a sufficiently high concentration of urea to reduce fouling of the resin.

2. A method of separating a biomolecule from an unclarified feedstock, said method comprising the steps of: a) solubilizing the unclarified feedstock in an aqueous urea solution to form a sample solution; b) passing the sample solution upwards through a bed of resin having affinity for the biomolecule, thereby binding the biomolecule to the resin and separating the biomolecule from the unclarified feedstock, wherein the sample solution comprises a sufficiently high concentration of urea to reduce fouling of the resin.

3. A method of separating a recombinant protein from an unclarified feedstock, said method comprising the steps of: a) solubilizing the unclarified feedstock in an aqueous urea solution to form a sample solution, said sample solution comprising between about 6.0 M and about 8.0 M urea; and b) passing the sample solution upwards through a bed of resin, thereby binding the recombinant protein to the resin and separating the recombinant protein from the fermentation broth or cell homogenate .

4. The method of Claim 3 wherein the sample solution comprises suspended whole cells.

5. The method of Claim 3 wherein the urea concentration in the sample solution is between about 6.3 M and about 7.0 .'

6. The method of Claim 5, additionally comprising the step of passing an aqueous urea wash solution upwards through the bed of resin with bound recombinant protein until the eluent is colorless.

7. The method of Claim 5, additionally comprising the step of passing an aqueous urea wash solution upwards through the bed of resin until substantially all of the non-bound components have eluted from the column.

8. The method of Claim 7 wherein the resin is an ion exchange resin.

9. The method of Claim 8 wherein the ion exchange resin is an anion exchange resin.

10. The method of Claim 9 additionally comprising the step of passing water or an aqueous salt solution through the ion exchange resin, thereby eluting the recombinant protein from the ion exchange resin.

11. The method of Claim 10 wherein the water or aqueous salt solution is passed downwards through the ion exchange resin.

12. The method of Claim 11 wherein sufficient water or aqueous salt solution is passed through the ion exchange resin to elute substantially all the recombinant protein from the ion exchange resin.

13. The method of Claim 12 wherein the recombinant protein is isolated from the eluent.

14. The method of Claim 8 wherein the ion exchange resin is a cation exchange resin.

15. The method of Claim 14 additionally comprising the step of passing water or an aqueous salt solution through the cation exchange resin, thereby eluting the recombinant protein from the cation exchange resin.

16. The method of Claim 15 wherein the water or aqueous salt solution is passed downwards through the cation exchange resin.

17. The method of Claim 16 wherein sufficient water or aqueous salt solution is passed through the ion exchange resin to elute substantially all the recombinant protein.

18. The method of Claim 17 wherein the recombinant protein is isolated from the eluent.

19. The method of any one of Claims 3 through 18 wherein the recombinant protein is formulated as a pharmaceutically acceptable drug product.

20. The method of Claim 13 or 18 wherein the recombinant protein isolated from the eluent is further purified and formulated as a pharmaceutically acceptable drug product.

21. The method of any one of Claims 3 through 18 wherein the recombinant protein is a fusion protein.

22. The method of Claim 21 wherein the fusion protein is cleaved such that an active biological protein is released^".

23. The method of Claim 22 wherein the active biological protein is further, purified and formulated as a pharmaceutically acceptable drug product.

24. The method of Claim 20 wherein the recombinant protein is selected from the group consisting of: GLP-1 (7-

37) OH, Exendin-3, Exendin-4, an analog of GLP-1 (7- 37) OH, or an analog of Exendin-4.

25. The method of Claim 24 wherein the recombinant protein is Val⁸-GLP-l(7-37)OH or Arg³⁴-GLP-1 (7-37) OH.

26. The method of Claim 23 wherein the active biologically active protein is selected from the group consisting of: GLP-1 (7-37) OH, Exendin-3, Exendin-4, an analog of GLP-1 (7-37) OH, or an analog of Exendin-4.

27. The method of Claim 26 wherein the biologically protein is Val⁸-GLP-l(7-37)OH or Arg³⁴-GLP-1 (7-37) OH.